Tumor vaccine

ABSTRACT

The invention relates to the fields of medicine, immunology, and oncology. More specifically, the invention relates to methods and compositions for inducing an immune response against a tumor in an animal subject. The invention provides that a lung cancer cell or other tumor cells, genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding an HLA antigen, and method for stimulating an immune response to a tumor with the tumor cell so genetically modified. The invention additionally provides a method of inhibiting a tumor, including a cancer such as lung cancer, by administering an allogeneic tumor cell, for example a cancer tumor cell such as a lung cancer tumor cell, genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding an HLA antigen.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 61/106,355, filed on Oct. 17, 2008.

FIELD OF THE INVENTION

The invention relates to the fields of medicine, immunology, andoncology. More specifically, the invention relates to methods andcompositions for inducing an immune response against a tumor in ananimal subject.

BACKGROUND OF THE INVENTION

Lung cancer is the most common cause of death due to cancer in theUnited States. For 2002, the American Cancer Society predicted thatalmost 170,000 new cases of lung cancer would be diagnosed and that155,000 people would die from the disease. Patients with locallyadvanced or metastatic non-small cell lung cancer (NSCLC) make up 70% ofthe newly diagnosed cases.

Current recommendations for patients with inoperable disease includeplatinum-based chemotherapy plus radiation therapy in locally advanceddisease, or chemotherapy alone in patients with metastases. Typicalresponse rates are between 15% to 30%, with median survivals of lessthan one year. Meta-analysis of 52 phase III clinical trials randomizingmetastatic NSCLC patients between best supportive care and chemotherapyconcluded that chemotherapy increases the chance of 1 year survival by10% and the median survival by 6 weeks. A recent report from the BigLung Trial group (BLT) reported similar results. The aggressiveness ofNSCLC is thought to relate to its ability to evade the immune systemperhaps by suppressing immune response priming by means of CD4regulatory cells and/or by producing immunosuppressive cytokines such asTGF-β.

Thus, there exists the need to develop effective therapies to treat atumor, including cancers such as lung cancer. The present inventionsatisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The invention provides a tumor cell, for example, a lung cancer cell orother tumor cells, genetically modified to express a nucleic acidencoding CD80 (B7.1) and a nucleic acid encoding an HLA antigen. Theinvention also provides a method of stimulating an immune response to atumor, including a cancer tumor such as a lung cancer tumor, byadministering an allogeneic lung cancer tumor cell genetically modifiedto express a nucleic acid encoding CD80 (B7.1) and a nucleic acidencoding an HLA antigen. The invention additionally provides a method ofinhibiting a tumor, including a cancer such as lung cancer, byadministering an allogeneic tumor cell, for example a cancer tumor cellsuch as a lung cancer tumor cell, genetically modified to express anucleic acid encoding CD80 (B7.1) and a nucleic acid encoding an HLAantigen. According to some embodiments of the invention, the vaccine isadministered more than once.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B shows flow cytometry analysis. Panel A: Quality controlof vaccine cells. Representative samples of vaccine cells coexpressingB7.1 (CD80) and HLA A1 (left panel) or HLA A2 (right panel) analyzed byflow cytometry. The percentage of double positive cells is indicated.CD80 and the HLA A allele must be coexpressed on 70% or more of thecells to qualify for immunization. Panel B. Patient CD8 cells purifiedfor ELI-spot assays. Flow cytometry of a representative sample ofpatient CD8 (right panel) cells purified by negative selection and usedfor ELIspot analysis; the purity of cells is given in %. Left panelshows isotype control.

FIG. 2 shows analysis of CD8 immune response: Immunization of advancedlung tumor patients generates strong CD8 response. The frequency ofIFN-y-spot forming CD8 cells obtained from lung tumor patients isplotted against the time on study in weeks. Immunizations were givenevery two weeks, zero representing the preimmunization status. 20,000purified CD8 cells were used for ELI-spot assays. Panel A: Frequency ofspot forming CD8 cells from HLA A1 and A2 positive patients challengedwith HLA A1 or A2 transfected (matched) AD 100 tumor cells at a ratio of20:1=CD8:AD100. Panel B: Frequency of spot forming CD8 cells from HLA A1positive patients challenged with A2-AD100 or HLA A2-CD8 cells werechallenged with A1-AD100 (mismatched). Panel C: Frequency of spotforming CD8 cells from non HLAA-1 or A2 patients cells challenged withA1 and A2 transfected AD100 (unmatched). Panel D: Frequency of spotforming CD8 cells from all patients challenged with untransfected wildtype (w. t.) AD100 or, Panel E, with K562. Panel F: Mean frequency ofspot forming CD8 cells from all patients challenged with any of theAD100 w. t. or transfected cells. Panel G: CD8 spot forming response ofindividual, clinically responding patients. The mean number of spotsafter restimulation with AD100 w. t., AD100-A1, AD100-A2, K562 ornothing in quadruplicate wells is plotted against time after studyentry. Arrows indicate the time of last immunization. Patient 1004,1007, 1010 contain follow up data analyzed at the points indicated aftercompletion of nine immunizations (18 weeks). HLA type of each patient isindicated in brackets.

FIG. 3 shows the median survival time of all patients at the time ofanalysis. The median survival time was 18 months, exceeding the expectedmedian survival time of less than one year for this group of patients.

FIG. 4 shows overall survival for the 19 B7 vaccine-treatednon-small-cell lung cancer study patients.

FIGS. 5A and B show analysis of CD8 immune response. FIG. 5A (top twopanels) shows CD8 prior to immunization or at 6,12 and 18 weeks afterchallenge with untransfected (AD wild type) vaccine cells or K562control. FIG. 5B (lower six panels) shows CD8 response after terminationof vaccination (arrow) in patients with clinical response.

FIG. 6 illustrates the sequence and annotation of one embodiment of aBPV-1-B7.1-HLA A1 vector.

FIG. 7 illustrates the survival curve from initiation of phase Iclinical trial to present or to last survivor.

FIG. 8 illustrates patient response to different levels of vaccination.Patients who received a second or third course of vaccination faredbetter in terms of both clinical response and survival.

DETAILED DESCRIPTION

The invention relates to the discovery that administering allogeneictumor cells expressing or caused to express CD80 (B7.1) and HLA antigensto cancer patients resulted in an anti-tumor immune response in thepatients. More particularly, CD8-mediated immune responses were elicitedin stage IIIB/IV NSCLC patients immunized several times with allogeneicNSCLC cells transfected with CD80 (B7.1) and HLA-A1 or A2. Immunizationsignificantly increased the frequencies of interferon-Y-secreting CD8 Tcells in all but one of the patients tested as discussed in more detail,below, in a clinical analysis of one set of patients, five of fourteenpatients responded to immunization with stable disease or partial tumorregression. Further characterization was performed with additionalpatients.

Carcinoma of the lung is the leading cause of cancer death and thesecond most commonly occurring cancer in both men and women in theUnited States (Jemal, et al., CA Cancer J. Clin. 53: 5-43 (2003).Non-small-cell lung cancers (NSCLC) are considered to be minimally ornonimmunogenic, and may contain CD4 regulatory cells that suppressgeneration of cytotoxic lymphocytes (CTL) (Woo, et al., J. Immunol. 168:4272-4276 (2002)). Although NSCLC has not been considered a goodcandidate for immunotherapy, the studies disclosed herein are based onthe hypothesis that NSCLC is indeed suitable for successful vaccinetherapy because the tumor cells have not been exposed to immune attackand have not yet developed resistance mechanisms.

Immunotherapy trials for lung cancer have previously yielded noconsistent benefit in humans (Ratto, et al., Cancer 78: 244-251 (1996);Lissoni, et al., Tumori 80: 464-467 (1994); Ratto, et al., J. Immunother23: 161-167 (2000)). Vaccine trials with B7.1 (CD80) transfectedallogeneic or autologous cells have not been reported in patients withNSCLC prior to the studies disclosed herein, although similar vaccineshave shown good activity in other human studies (Antonia, et al., J.Urol. 167: 1995-2000 (2002); Honig, et al., Cancer Immunol. Immunother.49: 504-514 (2000); Hull, et al., Clin. Cancer. Res. 6: 4101-4109(2000); von Mehren, et al., Clin. Cancer Res. 6: 2219-2228 (2000)). Theobjectives of the studies disclosed herein were to assess the safety,immunogenicity, and clinical response to an allogeneic whole cell tumorvaccine transfected with CD80 and HLA A1 or A2 administered to patientswith advanced metastatic NSCLC. Disclosed herein are results on vaccinesafety, clinical response, and overall survival.

As disclosed herein, to determine whether CD8 mediated immune responsescould be elicited in stage IIIB/IV NSCLC patients, initially fourteensubjects were immunized several times with allogeneic NSCLC cellstransfected with CD80 (B7.1) and HLA-A1 or A2. Additional patients wereadded. Patients enrolled were matched or unmatched at the HLA A1 or A2locus and their immune response compared. Immunization significantlyincreased the frequencies of interferon-y secreting CD8 T cells in allbut one patient in response to ex vivo challenge with NSCLC cells. TheCDS response of matched and unmatched patients was not statisticallydifferent. NSCLC reactive CD8 cells did not react to IL562. Clinically,five of fourteen patients responded to immunization with stable diseaseor partial tumor regression. The study demonstrates that CD8IFN-yresponses against non-immunogenic or immunosuppressive tumors can beevoked by cellular vaccines even at advanced stages of disease. Thepositive clinical outcome suggests that non immunogenic tumors may behighly susceptible to immune effector cells generated by immunization.

Thus, it has been discovered that the administration to a tumor patientof modified tumor cells expressing CD80 and an HLA antigen results indesirable therapeutic effects. Hence, in one embodiment, the inventionprovides a tumor lung cancer cell into which has been introduced a firstnucleic acid encoding CD80 and a second nucleic acid encoding HLAantigen. These modified tumor cells can be administered more than once.The modified tumor cells can be administered 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times. Preferably, the vaccineis administered between 2 and 9 times.

As used in this specification, the singular forms “a,” “an” and “the”specifically also encompass the plural forms of the terms to which theyrefer, unless the content clearly dictates otherwise. As used herein,unless specifically indicated otherwise, the word “or “is used in the“inclusive” sense of “and/or” and not the “exclusive” sense of”either/or.” In the specification and the appended claims, the singularforms include plural referents unless the context clearly dictatesotherwise.

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 20%. As used in this specification,whether in a transitional phrase or in the body of the claim, the terms“comprise (s)” and “comprising” are to be interpreted as having anopen-ended meaning. That is, the terms are to be interpretedsynonymously with the phrases “having at least” or “including at least”.When used in the context of a process, the term “comprising” means thatthe process includes at least the recited steps, but may includeadditional steps. When used in the context of a compound or composition,the term “comprising” means that the compound or composition includes atleast the recited features or components, but may also includeadditional features or components.

The term “tumor” is used to denote neoplastic growth which may be benign(e.g., a tumor which does not form metastases and destroy adjacentnormal tissue) or malignant/cancer (e.g., a tumor that invadessurrounding tissues, and is usually capable of producing metastases, mayrecur after attempted removal, and is likely to cause death of the hostunless adequately treated) (see Steadman's Medical Dictionary, 26th Ed,Williams & Wilkins, Baltimore, Md. (1995)).

The invention also provides a method of stabilizing or reversing a tumorload in a patient by administering to the patient an allogeneic tumorcell into which has been introduced a first nucleic acid encoding CD80and a second nucleic acid encoding an HLA antigen.

In another embodiment, the invention provides a tumor cell, which can bea tumor cancer cell such as a lung cancer cell, genetically modified toexpress a nucleic acid encoding CD80 (B7.1) and a nucleic acid encodingan HLA antigen.

Exemplary HLA antigens include, but are not limited to, HLA A1, HLA A2,HLA A3, HLA A27, and the like. In a particular embodiment, the HLAantigen can be HLA A1 or HLA A2 (see Examples). One of skill in the artwill appreciate that there are a number of different nucleic acidsequences encoding HLA antigens which may be used according to theinvention without departing from the same (see below). Any suitablematerials and/or methods known to those of skill can be utilized incarrying out the present invention. However, preferred materials andmethods are described. Materials, reagents and the like to whichreference is made in the following description and examples areobtainable from commercial sources, unless otherwise noted.

Technical and scientific terms used herein have the meaning commonlyunderstood by one of skill in the art to which the present inventionpertains, unless otherwise defined.

Reference is made herein to various methodologies and materials known tothose of skill in the art. Standard reference works setting forth thegeneral principles of recombinant DNA technology include for example,Ausubel et al., Current Protocols in Molecular Biology (Supplement 56),John Wiley & Sons, New York (2001); Sambrook and Russel, MolecularCloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, ColdSpring Harbor (2001); Kaufman et al., Eds., Handbook of Molecular andCellular Methods in Biology in Medicine, CRC Press, Boca Raton (1995);McPherson, Ed., Directed Mutagenesis: A Practical Approach, IRL Press,Oxford (1991). Standard reference works setting forth the generalprinciples of pharmacology include Goodman and Gilman's ThePharmacological Basis of Therapeutics, 10th Ed., McGraw Hill CompaniesInc., New York (2001). The compositions according to the invention areoptionally formulated in a pharmaceutically acceptable vehicle with anyof the well known pharmaceutically acceptable carriers, includingdiluents and excipients (see Remington's Pharmaceutical Sciences, 18thEd., Gennaro, Mack Publishing Co., Easton, Pa. 1990 and Remington: TheScience and Practice of Pharmacy, Lippincott, Williams & Wilkins, 1995).While the type of pharmaceutically acceptable carrier/vehicle employedin generating the compositions of the invention will vary depending uponthe mode of administration of the composition to a mammal, generallypharmaceutically acceptable carriers are physiologically inert andnon-toxic. Formulations of compositions according to the invention maycontain more than one type of compound of the invention), as well anyother pharmacologically active ingredient useful for the treatment ofthe symptom/condition being treated.

In some embodiments, the cancer cell can be a lung tissue cancer cell(also referred to as “lung cancer cell”) such as an adenocarcinoma celltype, for example, the lung cancer cell can be the AD 100 cell line, asexemplified hereinafter.

The invention additionally provides a method of stimulating an immuneresponse to a tumor, for example, a cancer such as a lung cancer, in apatient by administering an allogeneic tumor cell genetically modifiedto express a nucleic acid encoding CD80 (B7.1) and a nucleic acidencoding an HLA antigen. The tumor cell can be a cancer cell, forexample, a lung cancer tumor cell.

The methods of the present invention are intended for use with anysubject that may experience the benefits of the methods of theinvention. Thus, in accordance with the invention, “subjects”,“patients” as well as “individuals” (used interchangeably) includehumans as well as non-human subjects, particularly domesticated animals.

In one embodiment, a method of the invention can include matching theHLA antigen to the individual administered the tumor lung cancer cell.Methods of determining HLA haplotypes are well known to those skilled inthe art, for example, using well known serological assays usingantibodies to HLA alleles or the mixed lymphocyte reaction. In aparticular embodiment, a method of the invention can be performed withthe HLA antigen HLA A1, HLA A2, HLA A3 or HLA A27. The methods of theinvention cause various tumor cells (e.g., lung cancer cells) including,for example, an adenocarcinoma such as the AD100 cell line exemplifiedhereinafter.

In still another embodiment, the invention provides a method ofinhibiting a tumor by administering an allogeneic tumor cell geneticallymodified to express a nucleic acid encoding CD80 (B7.1) and a nucleicacid encoding an HLA antigen. The tumor can be, for example, a cancertumor cell such as a lung cancer tumor cell. In certain embodiments, thetumor inhibited is lung cancer by the administration of an allogeneiccancer cell modified to express CD80 (B7.1) and an HLA antigen.

As used herein, an “allogeneic cell” refers to a cell that is notderived from the individual to which the cell is to be administered,that is, has a different genetic constitution than the individual. Anallogeneic cell is generally obtained from the same species as theindividual to which the cell is to be administered. For example, theallogeneic cell can be a human cell, as disclosed herein, foradministering to a human patient such as a cancer patient. As usedherein, an “allogeneic tumor cell” refers to a tumor cell that is notderived from the individual to which the allogeneic cell is to beadministered.

Generally, the allogeneic tumor cell expresses one or more tumorantigens that can stimulate an immune response against a tumor in anindividual to which the cell is to be administered. As used herein, an“allogeneic cancer cell,” for example, a lung cancer cell, refers to acancer cell that is not derived from the individual to which theallogeneic cell is to be administered. Generally, the allogeneic cancercell expresses one or more tumor antigens that can stimulate an immuneresponse against a cancer in an individual to which the cell is to beadministered, for example, a lung cancer.

As used herein, a “genetically modified cell” refers to a cell that hasbeen genetically modified to express an exogenous nucleic acid, forexample, by transfection or transduction. A cell can be geneticallymodified to express, for example, a nucleic acid encoding CD80 (B7.1)and/or a nucleic acid encoding an HLA antigen, as disclosed herein. Whena cell is to be genetically modified to express more than onepolypeptide, for example, CD80 (B7.1) and an HLA antigen, it isunderstood that the polypeptides can be encoded on separate nucleicacids (see Example 1) or on the same nucleic acid, if desired. Methodsof genetically modifying a cell are well known to those skilled in theart.

The invention provides methods and compositions for stimulating animmune response in a cancer patient. The compositions and methods areparticularly useful for stimulating an immune response againstnon-immunogenic tumors. As used herein, a non-immunogenic tumor is atumor that does not elicit a spontaneous immune response detectable, forexample, by appreciable stimulation of CD8 T cells that produceinterferon-γ (IFNγ) in tumor infiltrating lymphocytes (TILs).

Traditionally, melanoma and other immunogenic tumors have been preferredfor treatment by immunotherapy. In the present invention,non-immunogenic tumors are considered good targets for activeimmunotherapy because the tumor cells have not been immuno-selected forevasion of the CTL response. Exemplary non-immunogenic tumors include,but are not limited to, lung, pancreatic, and the like.

A particularly useful nonimmunogenic tumor type is non small cell lungcancer (NSCLC), as exemplified herein. NSCLC tumors are good targets foractive immunotherapy because they are non-immunogenic and do notspontaneously generate CTL responses. Therefore, NSCLC tumor cells havenot developed evasive mechanisms towards cytotoxic T and natural killer(NK) cells, and NSCLC tumors are susceptible to cytotoxic attack. Asdisclosed herein, a composition of the invention was used tosuccessfully slow tumor growth in NSCLC patients (see Examples II andIII).

NSCLC tumors can also be genetically engineered to express and secretegp96 and enhance the effectiveness of a vaccine because it combinesadjuvant activity with polyvalent peptide specificity. Polyvalenceprevents immunoselection and evasion. Tumor secreted gp96 activatesdendritic cells (DC), natural killer cells (NK) and cytotoxic Tlymphocytes (CTL), activating innate and adaptive immunity. Tumor cellscan be killed by NK-specific mechanisms, by promiscuous killing of CD8CTL through NKG2D, and by MHC restricted CD8 CTL activity. Theactivation of DC and NK by tumor secreted gp96 may also counteract thegeneration of immuno-suppressive CD4 regulatory cells found in NSCLCtumors. Tumor secreted gp96 stimulates, antigen cross presentation viathe CD91 receptor on DC and macrophages. NSCLC are known to share tumorantigens also found in melanoma and may be endowed with additionalshared antigens. Therefore allogeneic, gp96 secreting tumor cells usedas vaccine are expected to generate immunity to the patient's autologoustumor. Similarly, a composition of the invention containing anallogeneic tumor cell expressing CD80 and an HLA antigen can generateimmunity to the patient's autologous tumor.

Lung tumors prevent priming of CTL by regulatory cells, by TGF-Psecretion and by down regulation of MHC class I. Therefore, immunogenicvaccines are needed to generate a CTL response. Lung tumors aresusceptible to CTL killing because they have not been selected for CTLevasion. Lung tumor TIL contain large numbers of CD4 regulatory cellssuppressing priming. In contrast, melanoma TIL contain antigen specificCD8 CTL whose killing activity has been blocked, indicating that priminghas taken place already. As disclosed herein, lung cancer patients weresuccessfully treated with a vaccine containing an allogeneic tumor cellgenetically modified to express CD80 (B7.1) and an HLA antigen (ExamplesII and III). Thus, immunotherapy (vaccine therapy) of NSCLC is usefulfor treating this otherwise deadly disease.

As disclosed herein, an adenocarcinoma is an exemplary lung cancer thatcan be used in compositions and methods of the invention to express CD80(B7.1) and an HLA antigen. Other types of lung cancer are well known,and cells derived from other types of lung cancers can be similarly usedin compositions and methods of the invention. Exemplary lung cancersinclude, for example, non-small cell lung cancer, which can beadenocarcinoma, squamous cell carcinoma, or large cell carcinoma, smallcell lung cancer, and carcinoids. One skilled in the art can readilyobtain tissue samples from various types of lung cancers and generate acell line useful for treating a lung cancer, using methods similar tothose disclosed herein. Similarly, other types of nonimmunogenic tumorscan be used to generate allogeneic tumor cells that can be geneticallymodified to express CD80 (B7.1) and an HLA antigen and used to treat asimilar type of tumor or a tumor expressing similar types of tumorantigens.

An exemplary allogeneic tumor cell is the AD 100 cell line, which is ahuman lung adenocarcinoma cell line, as disclosed herein. Other lungcancer cell lines are well known to those skilled in the art and can besimilarly used to generate an allogeneic cell genetically modified withCD80 (B7.1) and an HLA antigen. For example, numerous cell lines,including lung cancer cell lines are well known and available from theAmerican Type Culture Collection (ATCC; Manassas Va.). Exemplary NSCLCcell lines include, but are not limited to, NCI-H2126[H2126] (ATCCCCL-256); NCI-H23 [H23] (ATCC CRL-5800); NCI-H1299[H1299] (ATCCCRL-5803); NCI-H358 [H358] (ATCC CRL-5807); NCI-H810 [H810] (ATCCCRL-5816); NCI-H522 [H522] (ATCC CRL-5810); NCI-H1155 [H1155] (ATCCCRL-5818); NCI-H647 [H647] (ATCC CRL-5834); NCI-H650 [H650] (ATCCCRL-5835); NCI-H838[H838] (ATCC CRL-5844); NCI-H920 [H920] (ATCCCRL-5850); NCI-H969 [H969] (ATCC CRL-5852); NCI-H1385 [H1385] (ATCCCRL-5867); NCI-H1435[H1435] (ATCC CRL-5870); NCI-H1437[H1437] (ATCCCRL-5872); NCI-H1563[H1563] (ATCC CRL-5875); NCI-H1568[H1568] (ATCCCRL-5876); NCI-H1581[H1581] (ATCC CRL-5878); NCI-H1623[H1623] (ATCCCRL-5881); NCI-H1651 [H1651] (ATCC CRL-5884); NCI-H1693[H1693] (ATCCCRL-5887); NCI-H1703[H1703] (ATCC CRL-5889); NCI-H1734[H1734] (ATCCCRL-5891); NCI-H1755[H1755] (ATCC CRL-5892); NCI-H1770 [H1770] (ATCCCRL-5893); NCI-H1793[H1793] (ATCC CRL-5896); NCI-H1838[H1838] (ATCCCRL-5899); NCI-H1869[H1869] (ATCC CRL-5900); NCI-H1915 [H1915] (ATCCCRL-5904); NCI-H1944[H1944] (ATCC CRL-5907); NCI-H1975[H1975] (ATCCCRL-5908); NCI-H1993 [H1993] (ATCC CRL-5909); NCI-H2023[H2023] (ATCCCRL-5912); NCI-H2030 [H2030] (ATCC CRL-5914); NCI-H2073 [H2073] (ATCCCRL-5918); NCI-H2085 [H2085] (ATCC CRL-5921); NCI-H2087 [H2087] (ATCCCRL-5922); NCI-H2106 [H2106] (ATCC CRL-5923); NCI-H2110 [H2110] (ATCCCRL-5924); NCI-H2135 [H2135] (ATCC CRL-5926); NCI-H2172[H2172] (ATCCCRL-5930); NCI-H2228 [H2228] (ATCC CRL-5935); NCI-H2291 [H2291] (ATCCCRL-5939); NCI-H2342 [H2342] (ATCC CRL-5941); NCI-H2347 [H2347] (ATCCCRL-5942); NCI-H2405 [H2405] (ATCC CRL-5944); NCI-H2444 [H2444] (ATCCCRL-5945); and NCI-H2122 [H2122] (ATCC CRL-5985). These and other tumorcell lines, particularly those of nonimmunogenic tumors, can similarlybe used in compositions and methods of the invention.

As disclosed herein, these and other tumor cell lines can be geneticallymodified to express exogenous molecules that enhance an immune responseto tumor antigens. Such molecules include, but are not limited to, CD80(B7.1), human HLA antigens, for example, HLA A1, A2, A3, A27, and thelike. One skilled in the art can readily obtain appropriate sequencesencoding such molecules using well known methods. One skilled in the artwill readily understand that variants of such molecules are available orcan be readily obtained using well known methods. Based on knowncomplete or partial sequences, one skilled in the art can use well knownmolecular biology methods to obtain nucleic acid sequences suitable togenetically modify a tumor cell, as disclosed herein. It is understoodthat these exemplary sequences as well as natural variations of suchsequences are considered within the scope of the invention.

Exemplary nucleic acid sequences encoding molecules that enhance animmune response are available, for example, from GenBank, includingcomplete and partial cDNA sequences as well as genomic sequences, andsuch sequences can be used to obtain nucleic suitable nucleic acidsequences encoding desired immune enhancing molecules. A representativeselection of such sequences available from GenBank include, but are notlimited to, GenBank accession numbers NT_(—)005612; NM_(—)012092;NM_(—)175862; NM_(—)006889; NM_(—)005191; BC_(—)042665; NM_(—)012092;NM_(—)175862; NM_(—)006889; NM_(—)152854; NM_(—)005214; NM_(—)005514;NM_(—)002116; Z70315; NM_(—)002127; AH013634; L34703; L34734; AF389378;U30904; AH006709; AH006661; AH006660; X55710; U04244; U35431; M24043;U03859; NM_(—)005514; NM_(—)002116; Z30341; NM_(—)012292; NM_(—)002127;NM_(—)002117; AH007560; AH000042; AB048347; AB032594; AJ293264;AJ293263; AB030575 AB030574; AB030573; AF221125; AF221124; AH009136;X60764; AB032597; L17005; Y13267; AH003586; Z46633; Z27120; Z33453;Z23071; X02457; X57954; K02883; U21053; U04243; U18930; L36318; L36591;L38504; L33922; M20179; M20139; M24042; M15497; M31944; U04787; U01848;M27537; U11267; U03907; U03863; U03862; U03861; NM002116; L34724;L34723; L34721; L34737; L34701; Z97370; L15370; AH003070; M20179;M16273; M16272; M15497; M19756; M19757; NT008413, and the like.

The compositions and methods of the invention are useful for stimulatingan immune response against a tumor. Such immune response is useful intreating or alleviating a sign or symptom associated with the tumor.Such an immune response can ameliorate a sign or symptom associated witha lung cancer. As used herein, by “treating” is meant reducing,preventing, and/or reversing the symptoms in the individual to which acompound of the invention has been administered, as compared to thesymptoms of an individual not being treated according to the invention.A practitioner will appreciate that the compositions and methodsdescribed herein are to be used in concomitance with continuous clinicalevaluations by a skilled practitioner (physician or veterinarian) todetermine subsequent therapy. Hence, following treatment thepractitioners will evaluate any improvement in the treatment of thepulmonary inflammation according to standard methodologies. Suchevaluation will aid and inform in evaluating whether to increase, reduceor continue a particular treatment dose, mode of administration, etc.

The methods of the invention can thus be used to treat a tumor,including, for example, a cancer such as a lung cancer. The methods ofthe invention can be used, for example, to inhibit the growth of a tumorby preventing further tumor growth, by slowing tumor growth, or bycausing tumor regression. Thus, the methods of the invention can beused, for example, to treat a cancer such as a lung cancer. It will beunderstood that the subject to which a compound of the invention isadministered need not suffer from a specific traumatic state. Indeed,the compounds of the invention may be administered prophylactically,prior to any development of symptoms (e.g., a patient in remission fromcancer). The term “therapeutic,” “therapeutically,” and permutations ofthese terms are used to encompass therapeutic, palliative as well asprophylactic uses. Hence, as used herein, by “treating or alleviatingthe symptoms” is meant reducing, preventing, and/or reversing thesymptoms of the individual to which a therapeutically effective amountof a composition of the invention has been administered, as compared tothe symptoms of an individual receiving no such administration.

The term “therapeutically effective amount” is used to denote treatmentsat dosages effective to achieve the therapeutic result sought.Furthermore, one of skill will appreciate that the therapeuticallyeffective amount of the composition of the invention may be lowered orincreased by fine tuning and/or by administering more than onecomposition of the invention (e.g., by the concomitant administration oftwo different genetically modified tumor cells), or by administering acomposition of the invention with another compound to enhance thetherapeutic effect (e.g., synergistically). The invention thereforeprovides a method to tailor the administration/treatment to theparticular exigencies specific to a given mammal. As illustrated in thefollowing examples, therapeutically effective amounts may be easilydetermined for example empirically by starting at relatively low amountsand by step-wise increments with concurrent evaluation of beneficialeffect. The methods of the invention can thus be used, alone or incombination with other well known tumor therapies, to treat a patienthaving a tumor. One skilled in the art will readily understandadvantageous uses of the invention, for example, in prolonging the lifeexpectancy of a lung cancer patient and/or improving the quality of lifeof a lung cancer patient.

Current recommendations for NSCLC patients with locally-advancedinoperable disease (stage IIIB) include platinum-based chemotherapy plusradiation therapy, and chemotherapy alone for patients with metastases(stage IV) (Clinical practice guidelines for the treatment ofunresectable non-small-cell lung cancer; adopted on May 16, 1997 by theAmerican Society of Clinical Oncology, J. Clin. Oncol. 15: 2996-3018,1997). Results of these approaches are nevertheless poor, and theincrease in survival is limited. The largest meta-analysis published todate concluded that chemotherapy increases the chance of 1-year survivalby 10% and median survival by 6 weeks (Chemotherapy in non-small celllung cancer: A meta-analysis using updated data on individual patientsfrom 52 randomizsed clinical trials. Non-Small Cell Lung CancerCollaborative Group. BMJ 311: 899, 1995). A recent report from the BigLung Trial group (BLT) reported similar results (Stephens et al., Proc.Am. Soc. Clin. Oncol. 21: 2002 (abstract 1661)). In phase III clinicaltrials, patients with metastatic disease have a median survival of lessthan 1 year (Schiller, et al., N. Engl. J. Med. 346: 92-98 (2002)).

Two phase III trials showed that after failure of first-linechemotherapy, only 6% of patients receiving standard second-linechemotherapy could expect to respond, with median survival beingapproximately 6 months (Shepherd, et al., J. Clin. Oncol. 18: 2095-2103(2000); Fossella, et al., J. Clin. Oncol. 18: 2354-2362 (2000)). In theexperiments described herein, the group of patients had a very poorprognosis as a result of their relapsed or metastatic disease status,and most patients had been unsuccessfully treated with surgery,radiation, and/or palliative chemotherapy, resulting in a projectedsurvival of less than 6 months.

A vaccination approach such as that disclosed herein can be an effectivemeans of inducing immune response in patients with nonimmunogenictumors. There is evidence that NSCLC tumors contain tumor antigens(Yamazaki, et al., Cancer Res. 59: 4642-4650 (1999); Weynants, et al.,Am. J. Respir. Crit. Care Med. 159: 55-62 (1999); Bixby, et al., Int. J.Cancer 78: 685-694 (1998); Yamada, et al., Cancer Res. 63: 2829-2835(2003)). However, it has been thought that lung tumors are poorcandidates for immunotherapy because they are poorly immunogenic and arepotentially immunosuppressive (Woo, et al., J. Immunol. 168: 4272-4276(2002); Woo et al., Cancer Res. 61: 4766-4772 (2001); Neuner, et al.,Int. J. Cancer. 101: 287-292 (2002); Neuner, et al., Lung Cancer 34(supplement 2): S79-82 (2001); Dohadwala, et al., J. Biel Chem. 276:20809-20812 (2001)), thereby anergizing or tolerizing T-cells (Schwartz,J. Exp. Med. 184: 1-8 (1996); Lombardi, et al., Science 264: 1587-1589(1994)). Lung tumors, therefore, have not been subjected to immuneattack, and hence have not been able to evolve evasive mechanisms toresist immune effector cells. Lung tumors, unlike immunogenic tumorsthat harbor tumor-infiltrating lymphocytes, thus may succumb to killerCTLs, especially in light of the involvement of CD8 CTLs in tumorrejection in a number of model systems (Podack, J. Leukoc. Biel. 57:548-552 (1995)).

As disclosed herein, an allogeneic whole cell vaccine was chosen becausewhole cell. Vaccines have given the best clinical results so far. Forexample, statistically significant survival benefit occurred when awhole cell melanoma vaccine was administered (Morton, et al., Ann Surg.236: 438-449 (2002)). In contrast, vaccine directed at a single epitopemay have limited utility due to tumor escape mutants (Velders, et al.,Semin Oncol. 25: 697-706 (1998)). The additional advantage of a wholecell vaccine approach is that it does not require a priori delineationof specific lung tumor antigens. If vaccination is successful and CTLsare generated, as was found in the experiments disclosed herein, theresponsible antigenic sites can be identified later. Allogeneiccell-based vaccines offer a good alternative to autologous vaccinesunder the assumption that lung tumor antigens are shared in lung tumorsof different patients, and the antigens can be cross-presented by thepatients' antigen-presenting cells. Although there is only limitedevidence for shared antigens in lung tumors (Yamazaki, et al., CancerRes. 59: 4642-4650 (1999); Yamada, et al., Cancer Res. 63: 2829-2835(2003)), this has been shown in other tumors (Fong, et al., Annu. Rev.Immunol. 18: 245-273 (2000); Boon, et al., Annu. Rev. Immunol.12:337-365 (1994)).

To obtain direct evidence that the CD8 cells generated in response toallogeneic vaccination recognize autologous tumor cells, tumor specimensshould be obtained at the time of surgery. Tumor specimens were notavailable in the trial of patients disclosed herein with advanceddisease (see Examples I1 and 111). However, the prolonged maintenance ofa high frequency of patient CD8 cells reacting to AD100 in vitro, andtheir increase in some patients (No. 1004 and No. 1007; FIG. 5) evenafter cessation of external vaccination, is consistent with the immunestimulation of patient CD8 cells by the autologous tumor and theircross-reaction with the allogeneic vaccine.

In the experiments disclosed herein, although only one patient had apartial response, five other patients had stable disease. Enhancedimmune reactivity was demonstrated by a CD8-mediated tumor-specificimmune response. The fact that six (32%) of 19 patients with very poorprognosis exhibited disease stabilization of a rapidly lethal condition,with median survival of the whole cohort reaching 18 months despitefar-advanced disease, is encouraging. The results disclosed hereinindicate that tumor progression is slowed by vaccination, and that thiseffect occurs regardless of whether or not patients are allogeneic tothe HLA A1 or A2 locus of the vaccine. The findings also indicate thatindirect antigen presentation can be effective in promoting antitumoractivity and that allogeneic MHC molecules enhance the effect.

In the results disclosed herein, the vaccine was well tolerated and thepatients' quality of life was very good, thus improving patient outcome.Because this is an immunologic product, it was assumed that someimmune-mediated side effects would be anticipated. Probable examples ofsuch phenomena of expected tolerable side effects were, for example, thelocal erythema at the vaccination site in five patients, and the episodeof arthritic pain experienced by one patient (see Example 3).

A composition of the invention containing a tumor cell geneticallymodified to express CD80 and an HLA antigen can be combined with aphysiologically acceptable carrier useful in a vaccine by including anyof the well known components useful for immunization. The components ofthe physiological carrier are intended to facilitate or enhance animmune response to an antigen administered in a vaccine. Theformulations can contain buffers to maintain a preferred pH range, saltsor other components that present the antigen to an individual in acomposition that stimulates an immune response to the antigen. Thephysiologically acceptable carrier can also contain one or moreadjuvants that enhance the immune response to the antigen. Formulationscan be administered subcutaneously, intramuscularly, intradermally, orin any manner acceptable for immunization.

An adjuvant refers to a substance which, when added to an immunogenicagent of the invention such as tumor cell genetically modified toexpress CD80 and an HLA antigen, nonspecifically enhances or potentiatesan immune response to the agent in the recipient host upon exposure tothe mixture. Adjuvants can include, for example, oil-in-water emulsions,water-in oil emulsions, alum (aluminum salts), liposomes andmicroparticles, such as, polysytrene, starch, polyphosphazene andpolylactide/polyglycosides.

Adjuvants can also include, for example, squalene mixtures (SAF-I),muramyl peptide, saponin derivatives, mycobacterium cell wallpreparations, monophosphoryl lipid A, mycolic acid derivatives, nonionicblock copolymer surfactants, Quil A, cholera toxin B subunit,polyphosphazene and derivatives, and immunostimulating complexes(ISCOMs) such as those described by Takahashi et al. Nature 344: 873-875(1990). For veterinary use and for production of antibodies in animals,mitogenic components of Freund's adjuvant (both complete and incomplete)can be used. In humans, Incomplete Freund's Adjuvant (IFA) is a usefuladjuvant. Various appropriate adjuvants are well known in the art (see,for example, Warren and Chedid, CRC Critical Reviews in Immunology 8: 83(1988); Allison and Byars, in Vaccines: New Approaches to ImmunologicalProblems, Ellis, ed., Butterworth-Heinemann, Boston (1992)). Additionaladjuvants include, for example, bacille Calmett-Guerin (BCG), DETOX(containing cell wall skeleton of Mycobacterium phlei (CWS) andmonophosphoryl lipid A from Salmonella minnesota (MPL)), and the like(see, for example, Hoover et al., J. Clin. Oncol., 11: 390 (1993);Woodlock et al., J. Immunotherapy 22: 251-259 (1999)).

FIG. 6 illustrates the sequence and annotation of one embodiment of aBPV-1-B7.1-HLA A1 vector derived from a bovine papillomavirus type 1(BPV-1) vector. The vector was further engineered to contain twoexpression cassettes for expression genes under the CMV and theMetallothioneine promoter, respectively. The sequence of this vector isshown at the end of the specification.

The compositions and methods of the invention disclosed herein areuseful for treating a patient having a tumor. Although particularembodiments are exemplified with lung cancers, it is understood that asimilar approach can also be used to treat other types of tumors,including cancers, using suitable allogeneic cells.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention. While the claimed invention has beendescribed in detail and with reference to specific embodiments thereof,it will be apparent to one of ordinary skill in the art that variouschanges and modifications can be made to the claimed invention withoutdeparting from the spirit and scope thereof. Thus, for example, thoseskilled in the art will recognize, or be able to ascertain, using nomore than routine experimentation, numerous equivalents to the specificsubstances and procedures described herein. Such equivalents areconsidered to be within the scope of this invention, and are covered bythe following claims.

Example 1 Allogeneic Vaccination with a B7.1/HLA-A Gene-ModifiedAdenocarcinoma Cell Line in Patients with Advanced Non-Small-Cell LungCancer

This example describes the protocol used for allogeneic vaccination witha B7.1 HLA-A gene-modified adenocarcinoma cell line in patients withadvanced non-small-cell lung cancer (NSCLC). This example describes theexperimental protocol used.

The following experiments were designed (a) to measure whether CD80 andHLA A transfected, allogeneic lung tumor cells used for immunotherapycan elicit tumor specific CD8-CTL activation and expansion, assessed byELIspot for IFN-γ; (b) to evaluate the safety and toxicity ofadministering allogeneic tumor cell vaccines transfected with B7.1 andHLA A1 or A2 in patients with Non-Small Cell Lung Carcinoma (NSCLC); and(c) to evaluate the antitumor effect of this B7.1 vaccine in clinicaloutcomes for patients with NSCLC.

Selection of Patients. Initially, fifteen patients with newly diagnosedor relapsed metastatic non-small cell lung cancer (NSCLC) were treated.The analysis of these 15 patients is described in Example 2. Anadditional four patients were added, for a total of 19 patients, and thefurther results with the 19 patients are described in Example 3. Thepatients had already failed chemotherapy, radiotherapy, surgery or acombination of all. Eligibility criteria were as follows: age>18 years,Eastern Cooperative Oncology Group (ECOG) performance status 0-2,measurable disease, signed informed consent, and histologicallyconfirmed NSCLC (stage IIIB with malignant pleural effusion, stage IV,or recurrent). Patients with brain metastasis were included if thesewere already treated. Patients were not eligible for study if they werereceiving chemotherapy, radiation therapy or a biologic modifying agentor during the preceding 4 weeks. All patients were treated in theoutpatient clinic at Sylvester Comprehensive Cancer Center/University ofMiami. A complete history and physical exam was performed, includingweight and vital signs, with performance status assessed by ECOGcriteria. The following tests were performed prior to enrollment:complete blood count; platelet count; chemistries (uric acid, calcium,phosphorus, transaminases including serum glutamic-oxaloacetictransaminase (SGOT) and serum glutamic-pyruvic transaminase (SGPT),alkaline phosphatase, lactate dehydrogenase (LDH), total and directbilirubin, blood urea nitrogen (BUN), creatinine, albumin, totalprotein, electrolytes, and glucose); and electrocardiogram (EKG). HLAtyping was obtained. Patients were followed twice monthly while beingvaccinated, with tumor response assessed by computed tomography (CT)scans. Tumor measurements were obtained from the results of radiographicstudies, including CT scans of relevant sites.

Vaccine Cell Line and Genetic Modification. A human lung adenocarcinomacell line was established in 1994 by Dr. N. Savaraj (University ofMiami, Department of Medicine) from a biopsy of a lung cancer patient,designated as AD100. The patient was a 74 year old white male whopresented in 1993 with initial symptoms of pelvic pain from bone erosionof the iliac crest due to metastatic pulmonary adenocarcinoma. Cancercells for culture were obtained by bone marrow aspiration from the areaof pelvic bone destruction. The patient was treated with radiationtherapy to the pelvis, but expired one month after diagnosis. The cellline derived from this patient has been kept in culture in standardmedium (described below) and is free of contamination by mycoplasma,virus or other adventitious agents. The cell line is homogeneous,adherent to plastic, and grows with a rate of division of approximately26 h.

Genetic Modification. AD100 was transfected with plasmid cDNA,pBMG-Neo-B7.1 and pBMG-His-HLA A2 or with B45-Neo-CM-A1-B7.1 (Yamazakiet al., Cancer Res., 59: 4642, 1999) Transfected cells were selectedwith G418 and histidinol. Verification of correct sequences was based onrestriction analysis and the expression of the relevant gene products,namely G418 or histidinol resistance for the vector sequence, HLA A1,A2, and B7.1 expression for the transfected cDNA. The cells wereirradiated to prevent their replication, for example, with 12,000 Radsin a cobalt (Co) irradiator, and stored frozen in 10% DMSO in aliquotsof 5×107 cells until use. Upon replating in tissue culture the cellsappeared viable for about 14 days but were unable to form colonies,indicating their inability to replicate. They were therefore consideredsafe for use as vaccine cells. The minimum requirement for their use asvaccine was the co expression of HLA A1 or A2 plus B7.1 on at least 70%of the cells, as shown in FIG. 1A for representative batches of vaccinecells. The untransfected AD100 line was negative by FACS for stainingwith anti HLA A1 or A2 or B7.1. FIG. 1A shows the quality control byflow cytometric analysis of CD80 and HLA A1 or A2 transfected AD100vaccine cells used for immunization.

Immunizations. Intracutaneous injections were given at multiple bodysites to reduce the extent of local skin reactions. Patients who wereHLA A1 or A2 received the corresponding HLA-matched vaccine, whereaspatients who were neither HLA A1 nor HLA A2 received HLA A1-transfectedvaccine (that is, HLA-unmatched vaccine). On a given vaccination day,the patient received the total dose of 5×107 irradiated cells (12,000rad) divided into two to five aliquots for administration as two to fiveintradermal injections of each aliquot in an extremity, spaced at least5 cm at needle entry from the nearest neighboring injection. A total ofnine immunizations (4.5×108 cells) were given over the course oftherapy, one every two weeks, provided that no tumor progressionoccurred under therapy (Table 1). On subsequent vaccinations, theinjection sites were rotated to different limbs in a clockwise manner.One course of vaccination comprised three biweekly injections. Patientswith evidence of stable disease or responding NSCLC by imagingevaluation (CT Scans) and none to moderate toxicity (grade<2) weretreated with an additional course at the same dose. The second course ofinjections started two weeks after the third vaccination that completedthe first course. In the absence of tumor progression by CT scans andwith no severe or life-threatening toxicity (grade>3), a third course atthe same dose of therapy was given, starting two weeks after the thirdvaccination of the second course of therapy. Clinical” toxicity, andimmunologic evaluations by blood tests prior to and after each coursewere performed was done. Patients were followed clinically weekly duringthe study, including monitoring blood counts and basic chemistries(Table 1).

Table 1 shows the treatment and evaluation schedule of NSCLC (IIIB/IV)patients. Patients were immunized nine times in biweekly intervals, asdiscussed above. Immunological assays were done prior to and after eachof three immunizations.

TABLE 1 Immunizations and Immunological Evaluations Study Entry Course 1Course 2 Course 3 Weeks on Study 1 2 4 6 7 8 10 12 13 14 16 18 19Pre-Entry Evaluation x Immunization # 1 2 3 4 5 6 7 8 9 ClinicalEvaluation x x x x x x x x x x x x x Toxicity Evaluation x x x x x x x xx x x x Immunological Evaluation # 1 2 3 4

Immunological Testing Immunological tests were performed included skintests delayed-type hypersenstivity (DTH) and enzyme-linked immunospot(ELISPOT) assays for interferon-γ IFN-γ. Immune responses mediated byCD4 cells were examined by DTH-reaction following intradermal injectionof 105A1, A2 or untransfected AD100-B7 vaccine cells. Purified CD8 cellswere obtained from patients prior to and after each course of threeimmunizations. CDS cells. Were enriched by negative depletion withanti-CD56, anti-CD4 and other antibodies using the Spin-sep prep (StemCell Technologies; Vancouver, Canada). Purity was better than 80% (FIG.1B) the primary contaminating cells being B cells (not shown). CD8 cellswere frozen in 10% dimethylsulfoxide (DMSO) and 20% fetal calf serum(FCS) containing medium for analysis until all vaccinations of a studypatient were completed. Analysis for pre-immune and post-vaccinationELISPOT frequency was carried out on the same day in the same microtiter plate. Assays were done in quadruplicate, stimulating 2×104purified patient CD8 cells with, respectively, 103 A1 or A2 transfectedor untransfected AD100, with K562 or with media only for three days anddetermining the frequency of IFN-y producing cells by ELISPOT. Immuneassays were performed prior to immunization and after 3, 6, and 9immunizations.

Statistical Analysis. Patient characteristics are presented as countswith percentages, or as mean values and range. Overall survival,estimated by the Kaplan-Meier product-limit method, is defined as timefrom enrollment onto study until death from any cause. In the absence ofdeath, follow-up was censored at the date of last patient contact.Univariate and multivariate proportional hazards regression were used todetermine whether patients' survival time was related to age(continuous), sex, race (other versus white non-Hispanic), tumorpathology (adenocarcinoma versus other), and HLA-matching of vaccine.Logistic regression was used for the corresponding analyses of clinicalresponse. For hazard ratios and the percentage of patients surviving,90% confidence intervals (CIs) L₉₀-U₉₀ are reported. These can beinterpreted as providing 95% confidence that the parameter beingestimated, such as the hazard ratio, exceeds L₉₀.

Example 2 Specific CD8 T Cell Response of Advanced Lung Cancer Patientsto Whole Cell Immunization with an Allogeneic Vaccine

This example describes the results of a 15 patient group study on wholecell immunization with an allogeneic vaccine.

Patients with advanced NSCLC stage IIIB/IV were HLA typed. HLA A1positive patients received the AD-A1-B7 vaccine; HLA A2 positivepatients received the AD-A2-B7 vaccine; and patients that were neitherHLA A1 nor A2 positive received either the AD-A1-B7 or AD-A2-B7 vaccine.The frequency of IFN-y secreting CD8 cells was determined by ELISPOTafter restimulation of purified patient-CD8 cells in vitro with HLA A1or A2 transfected or untransfected AD100. Controls included stimulationwith K562 and incubation of CD8 cells without stimulator cells.

ELISPOT responses of immunized tumor patients are presented as HLAmatched responses (FIG. 2A), representing the number of IFN-γ secretingCD8 cells obtained from HLA A1 or A2 patients challenged in vitro forthree days with HLA A1 or A2 transfected AD100 cells, respectively. HLAmismatched responses indicate the number of spots formed when CD8 cellsfrom A1 or A2 patients were challenged with A2 or A1 transfected AD100,respectively (FIG. 2B). The matched response increased 15-fold, from 6±4(standard error of the mean, SEM) IFN-γ secreting, pre-immune CD8 cells(per 20 thousand) to maximal 90±35 (SEM) IFN-γ secreting cells after siximmunizations and remained at this level during the next threeimmunizations. The mismatched response increased 5.7 fold, from 24±18 to142±42 maximal. Included in this group of nine patients is the onepatient who showed no response (0 spots) before or after threeimmunizations, at which time the tumor progressed and the patient wastaken off trial.

The remaining 5 patients were negative for HLA A1 or A2. These patientsCD8 response to challenge with A1 or A2 transfected AD 100 is shown asunmatched response in FIG. 2C. The frequency of IFN-γ secreting CDScells increased 21-fold from 4.8±1.8 pre-immune to 105±24 after threeimmunizations and stayed constant throughout the trial. This increase infrequency is similar to that of all patients' CD8 cells when challengedwith the untransfected wild type AD100 (FIG. 2D). Finally, thespecificity of the response is evident from the absence of an increaseof the response to K562 (FIG. 2E) or of unchallenged CD8 cells. The CD8response to K562 and to AD100 in its w. t. form or after geneticmodification is significantly different at each time point aftervaccination (FIG. 2F).

The CD8 response listed in Table 2 reports the response to the matchedvaccine for A1 or A2 positive patients. For non A1, A2 patients, it isthe response to AD100-A2. One of 15 patients could not be analyzed dueto renal failure unrelated to the trial prior to completing the firstcourse of immunization. Of the fifteen patients treated, five patientshad clinical responses: one partial response (PR), and four patientswith stable disease (SD). Four of these patients with clinicalresponses, (PR+3 SD), are still alive with stabilization of theirdiseases without further therapy for: 31, 28, 25, and 12 months.

The patient that died, originally had SD for 5 months then progressedand died 15 months later in spite of several courses of palliativechemotherapy. In contrast, nine of the other ten patients that did notrespond to the vaccination are deceased except one patient who achievedstable disease after therapy with Iressa™. Table 2 summarizes the datafor all patients, including pre-trial treatment, clinical response toimmunization and immune response. Patients that had progressive diseasewhile under treatment went off study as indicated in Table 2.

Table 2 shows a summary of clinical responses, immunological CD8responses, survival and pretreatment of fifteen patients with advancedstage IIIB/IV NSCLC treated with allogeneic B7/HLA A transfected NSCLCvaccine. The abbreviations in Table 2 are: PD—progressive disease;NE—not evaluable for immune response, but included in survival analysison the right; PR—partial response; SD-sable disease; C—chemotherapy;R—radiation; S—surgery. Survival indicates time of survival since studyentry; +indicates patient alive; n. d. no done, patients off studybecause of progression.

TABLE 2 Summary of Clinical Responses, Immunological CD8 Responses,Survival and Pretreatment of Fifteen NSCLC Patients. Ifn-γ producing CD8cells to AD100-HLA Fold Time to challenge (spots per 20,000) Patient #Titer Previous Survival Progression Pre- 1st 2nd 3rd HLA Responseincrease TX (mos) (mos) immune course course course 1005 A1 PD 190 C + R10 — 0 190 n.d. n.d. 1012 A1 NE NE C 15 — 0.2 n.d. n.d. n.d. 1001 A2 PD25 C + S 18 — 0 25 n.d. n.d. 1002 A2 PD 1.6 C + S 22 — 41 65 n.d. n.d.1009 A2 PD 6.5 C  3 — 2 13 n.d. n.d. 1010 A2 PR 41 S   27+ 3 3.8 46 88157  1011 A2 PD 19 C 11 — 3 30 57 n.d. 1013 A2 PD 34 C + R + S  2 — 5.2164 178 n.d. 1014 A2 SD 19 C + S   13+ 3 1.6 30 30 25 1015 A2 PD 0 C + R 7 — 0 0 nd nd 1003 non SD 134 S   31+ 26+ 1 134 113 84 1004 non SD 424C + R 23 11   0 424 232 >450  1006 non PD 9.3 C + S   30+ — 16 150 n.d.n.d. 1007 non SD 14 C + R + S   29+ 23+ 1.2 2.8 .8 0/17 1008 non PD 32 C 6 — 5.6 178 n.d. n.d.

Five patients had a clinical response and the frequency of IFN-spotforming CD8 cells increased upon successive imnunization as measured bychallenge ex vivo with transfected or untransfected AD100, while thereactivity to K562 remained low and unchanged (FIG. 2E). In three of theclinically responding patients (FIG. 2; 1004, 1007, 1010), blood sampleswere obtained after completion of the 18 week treatment period at 35 to75 weeks post trial entry and showed still a considerable titer of CD8cells responding to AD100 (FIG. 2G). Indeed, in two of two patients(1004, 1007), the titer increased further even after immunization wasended at 18 weeks.

The median survival time of all patients at the time of analysis was 18months, exceeding the expected median survival time of less than oneyear for this group of patients (FIG. 3). 90% confidence intervals areshown in FIG. 3. Analysis of survival by MHC matching and by clinicalresponse revealed that HLA unmatched patients showed a survivaladvantage that with p=0.07 was not statistically significant whileclinical responders had a significant (p=0.008) survival advantage whencompared to non responders.

Safety. None of the 15 patients entered into the trial experienced anytreatment related serious adverse events, defined as deaths or eventsrequiring hospitalization. Treatment related side effects consisted oflocal erythema and swelling that resolved in three to four days. Onepatient complained about transient arthralgias that may have beentreatment related. One patient died within 30 days of the lastimmunization due to pulmonary failure; one patient who had previousepisodes of pericarditis experienced pericardial effusion during thelast course of immunization, requiring a pericardial window. No tumorcells were detected in the fluid; the patient responded to immunizationand is still in stable disease. As mentioned above, one patient hadrenal failure prior to completion of one course of immunization. None ofthese events were deemed likely to be treatment related by anindependent safety monitoring board.

Example 3 Further Characterization of Advanced Lung Cancer Patients toWhole Cell Immunization with an Allogeneic Vaccine

This example describes a continuation of the study described in Example2, including additional patients and time of study. Experiments wereperformed essentially as described in Example 2 and Raez et al., J.Clin. Oncol. 22: 2800-2807 (2004).

Patient Characteristics. The characteristics of the 19 study patientsare outlined in Table 3. Eastern Cooperative Oncology Group performancestatus was 0 to 1 in 18 patients (74%). Thirteen patients receivedvaccine matched for HLA, either A1 (three patients) or A2 (10 patients),whereas the six patients who were non-A1 and non-A2 received unmatchedvaccine (that is, HLA-A1 vaccine). While HLA A matched patients may beable to mediate CD8 responses by direct antigen presentation by thevaccine cells, it was reasoned that unmatched patients may, nonetheless,mount a CD8 response via indirect antigen presentation after vaccinecell death and antigen uptake by antigen presenting cells. Before beingenrolled on study, all patients had been previously treated: nine (47%)with surgery, six (32%) with radiation therapy, and 17 (89%) withchemotherapy. Among the chemotherapy-treated patients, 10 (53%) had beenunsuccessfully treated with more than one chemotherapy regimen.

TABLE 3 Characteristics of the 19 patients enrolled in the study.Characteristic No. of Patients Age, years* <50 2 50-59 6 60-69 5 70+ 6Sex Female 12 Male 7 Race/ethnicity White non-Hispanic 13 White Hispanic5 Black non-Hispanic 1 Pathology Adenocarcinoma 11 Bronchoalveolar 3Squamoous cell 3 Undifferentiated 2 Metastasis site Adrenal 1 Brain 3Liver 1 Lung 9 Pleura 1 Multiple sites† 4 ECOG performance status 0 4 114 2 1 HLA A1 3 A2 10 Neither 6 Abbreviation: ECOG, Eastern CooperativeOncology Goup. *Mean = 62 years; range 36 to 82 years. †Onepancreas/lung/adrenal; one brain/lung; one lung/adrenal; oneliver/lung/T-spine.

Clinical Outcomes. Eighteen patients received a total of 30 courses ofvaccine, 90 vaccinations in total (Table 4). Five patients receivedthree full courses, and two patients had two full courses. With theexception of one patient taken off study because a serious adverse event(SAE) occurred after the first vaccination (zero courses completed), theremaining 11 patients had one full course, after which they were takenoff study because of disease progression. Four patients experienced SAEsafter vaccination, none of which was judged to be vaccine-related.

TABLE 4 Outcomes in the 19 Patients Enrolled On Study. Outcome No. ofPatients Courses of vaccine received 0 1 1 11 2 2 3 5 Clinical responseComplete 0 Partial 1 Stable disease 5 Progressive disease 13 Serious AEs(grade 3 and 4) Pericardial effusion 2 Renal Failure 1 Respiratoryfailure 1 AEs (grade 1 or 2) Rash 1 Chest pain* 1 Joint pain 1 Status†Alive 7 Dead 12 Abbreviation: AE, adverse event. *Chest pain/shortnessof breath. †Alive: median follow-up was 36 months (range, 10 to 40months); time of death ranged from 1 to 23 months after entry on study.

During the first course of vaccination, a 58-year-old woman developedmalignant pericardial effusion requiring a pericardial window; thepatient was taken off study, discharged to hospice, and died 1 weeklater. She had previously been treated unsuccessfully with five lines ofpalliative chemotherapy before enrollment on study. A 76-year-old malepatient also developed a pericardial effusion requiring a pericardialwindow, but review of prior scans revealed developing pericardialeffusion before entry on study. This patient, who had received threecourses of vaccine before the SAE developed, continues to have stabledisease. He is currently alive and well after 31 months without anyfurther therapy.

A 55-year-old male was found to have worsening of chemotherapy-inducedrenal dysfunction the day of his first vaccination after he had alreadysigned consent 1 week earlier and underwent a preliminary skin test. Hisrenal function continued deteriorating in the following days, and hedied 3 months later. The fourth patient who experienced a SAE was a56-year-old woman with brain metastasis. During her second course ofvaccination, she developed respiratory failure, was then taken offstudy, and died within 30 days from progression of her disease. Thispatient had previously been unsuccessfully treated with four lines ofpalliative chemotherapy.

Regarding other side effects, one patient complained of transient painat the injection site. Four patients developed some erythema at thevaccination site that resolved within a week. One patient experiencedmoderate arthritic pain in several joints after the first course. We didnot find any patients with significant alteration of their laboratoryparameters, including: complete blood and platelet counts,creatinine/BUN, calcium, and liver function tests. Table 5 shows time toresponse, duration of response, and survival time for the six patientswho had response on study.

TABLE 5 Time to Response, Duration of Response, and Survival Time forthe Six Patients Who Had Response on Study. Time to Duration of SurvivalResponse Response Time Patient ID Response (months) (months) (months)*1010 PR 2.3 13   36+ 1003 SD 1.9 39+  40+ 1004 SD 1.6 3.5 23   1007 SD2.1 2.5 37+ 1014 SD 2.3 3.5 21+ 1016 SD 1.9 1.6 11+ Abbreviations: PR,partial response; SD, stable disease *Patients alive as of February,2004 denoted by plus sign.

One patient had a partial response lasting 13 months, and five showedstable disease ranging from 1.6 to 39+ months (Table 5). The clinicalresponse rate was 32% (six of 19 patients). As of February 2004, thesepatients had survival times ranging from 23 to 40+ months, and fivepatients were still alive.

After the patient who had a partial response developed new malignantlesions, verified by positron emission tomography scan, she was putunder observation for 2 months because her disease was judged clinicallynonaggressive. Several lesions subsequently decreased in size ordisappeared. This patient continues to have stable disease without needof palliative chemotherapy 36 months after completing vaccination. Onlyone of the six patients who had a response on treatment requiredsubsequent palliative chemotherapy. The remaining five patients continueto have stable disease without need of further treatment.

Among the other 13 patients who did not respond to therapy, only twowere alive as of February 2004. One of these patients experienceddisease stabilization with gefitinib (Iressa™), and the other isundergoing palliative chemotherapy.

Logistic regression analyses of age, sex, race. pathology, andHLA-matching of vaccine showed that none of these factors werestatistically significantly related (P>0.10 in all instances) toclinical response (that is, to partial response or stable disease).

FIG. 4 shows the Kaplan-Meier estimate of overall survival for the 19study patients (vertical tick marks indicate censored follow-up). Theestimated median survival time is 18 months (90% CI, 7 to 23 months).Estimates of 1-year, 2-year, and 3-year overall survival are 52% (90%CI, 32% to 71%), 30% (90% CI, 11% to 49%), and 30% (90% CI, 11% to 49%),respectively. As of February 2004, death had occurred in 12 patientsfrom 1 to 23 months after entry on study (Table 2). For the sevenpatients who are still alive, follow-up from study entry currentlyranges from 10 to 40 months, with a median follow-up time of 36 months.

Univariate proportional hazards regression analysis suggested a possiblyhigher mortality rate in patients receiving HLA-matched vaccine (hazardratio=4.5; 90% CI, 1.1 to 17.2), and a possibly lower mortality rate inpatients with adenocarcinoma (hazard ratio=0.3; 90% CI, 0.1 to 1.0). Amultivariate analysis involving five covariates (HLA-matching, age, sex,race, pathology), however, discounted an adverse effect of HLA-matchingof vaccine on overall mortality; the corresponding adjusted hazard ratiowas 1.9 (P=0.51). The adjusted hazard ratio for adenocarcinoma versusother pathologies was 0.2 (P=0.11), which is within the realm of chanceat conventional levels of significance.

Immune Response to Vaccination. This cohort of patients had been heavilypretreated and carried large tumor burdens that are believed to beimmunosuppressive. It was important, therefore, to establish whether thetumor vaccination protocol was able to induce a specific immune responsein these patients. Since the CD8 CTL response is thought to be criticalfor tumor rejection, studies were focused on this arm of the immunesystem. To distinguish between nonspecific natural killer (NK) activityand CD8 CTL activity, a two-fold strategy was employed. First, CD8 cellswere purified to eliminate NK cells by including anti-CD56 in thenegative selection cocktail of antibodies. Second, the CD8 cells werechallenged with K562, an NK target. NK contamination would result inhigh titers of cells responding to K562 challenge.

All but one patient had a measurable CD8 response after 6 weeks (threevaccinations) that tended to increase after 12 weeks and stabilize by 18weeks (Table 6). In vitro challenge of patient CD8 cells with wild typeA1 or A2 transfected AD100 did not reveal significant differences. Twopatients (patient Nos. 1012 and 1019) could not be evaluatedimmunologically because there was no follow-up sample available foranalysis due to early disease progression or adverse events. One patienthad only a very modest response, while most other patients showed astrong, highly statistically significant response to vaccination (seepre- and postimmunization titers on challenge with vaccine cells, andlack of response to K562 control; FIG. 5, top panels). All but onepatient had a measurable CD8 response after 6 weeks (three vaccinations)that tended to increase after 12 weeks and stabilize by IS weeks (Table6). In vitro challenge of patient CD8 cells with wild type A1 or A2transfected AD100 did not reveal significant differences. Two patients(patient Nos. 1012 and 1019) could not be evaluated immunologicallybecause there was no follow-up sample available for analysis due toearly disease progression or adverse events. One patient had only a verymodest response, while most other patients showed a strong, highlystatistically significant response to vaccination (see pre- andpostimmunization titers on challenge with vaccine cells, and lack ofresponse to K562 control; FIG. 5, top panels).

TABLE 6 CDB Response of Vaccinated Patients Immune Response of CDB Cellsto Vaccination* 0 Weeks 6 Weeks 12 Weeks 18 Weeks HLA/Patient AD- AD-AD- AD- AD- AD- AD- AD- AD- AD- AD- AD- NO. wt A1 A2 K562 wt A1 A2 K562wt A1 A2 K562 wt A1 A2 K562 A2/1001 4 6.2 0 2.6 51 49 25 6 A2/1002 12 1941 170 30 55 65 96 NO/1003 1 1 7 0 70 134 53 0 31 113 27 0 49 84 23 6NO/1004 0 0 0 5 321 424 195 0 216 232 150 0 283 450 130 0 A1/1005 15 0 040 92 190 80 34 NO/1006 13 17 12 11 156 152 132 16 NO/1007 0 1 0 0 0 3 00 1 1 1 0 0 0 2 0 NO/1008 5 6 4 10 97 180 48 3 A2/1009 3 4 2 17 13 39 1318 A2/1010 8 8 4 14 48 87 46 5 120 163 88 8 185 241 157 17 A2/1011 14 203 15 80 150 30 12 88 226 57 4 A2/1013 18 150 5 0 155 300 164 3 175 154178 3 A2/1014 3 2 2 10 28 20 30 9 30 20 30 12 25 23 25 4 A2/1015 0 0 0 00 0 0 0 A1/1016 138 120 128 4 144 150 163 5 127 120 164 15 A2/1017 0 110 4 100 200 200 3 NO/1018 13 44 0 9 51 200 52 9 NOTE. CD8 cellschallenged at a ratio of 20:1 = CD8: tumor cell. The mean spot number ofquadruplicate values is given. Abbreviations: AD-wt, AD100untransfected; AD-A1 or AD-A2, AD100 transfected with HLA A1 or A2; HLANO, No HLA A1 or A2. *Values are number of interferon-gamma secretingcells (spots per 20,000 CD8 cells) after in vitro challenge.

There was no statistically significant difference in the CD8 responsedepending on whether or not the patients were HLA-matched to the vaccine(Table 6). Most patients before vaccination had only low or absentimmune response to vaccine cells, and equally low activity to challengewith K562. One patient (No. 1016) had strong prevaccination CD8 activitytoward AD100 and only minimal activity toward K562 (FIG. 5, last panel),suggesting preexisting immune activity toward the tumor. Another patient(No. 1002) had high prevaccination K562 reactivity of his CD8 cells andlow activity toward AD100. Vaccination increased reactivity toward AD100and tended to decrease CD8 reactivity toward K562 when it was present.

The immune response of the six clinically-responding patients (FIG. 5B,lower panels) shows that CD8 titers to AD100 stimulation continue to beelevated up to 150 weeks after cessation of vaccination.

Given the advanced stage of disease in patients enrolled in the studiesdisclosed herein, the evidence of some clinical benefit was unexpectedand encouraging. Moreover, since the B7-vaccine tested here induced CD8CTL responses, it may be that the CD8 response is causally related tothe clinical outcome seen here. Additional studies are performed in thesetting of minimal disease. Patients with early stage NSCLC (stageI/II)are vaccinated after surgery to decrease the chance of relapse andpotentially prolong survival.

The results described in this example show that tumor progression can beslowed by vaccination and that this effect occurs regardless of whetheror not patients are allogeneic to the HLA A1 or A2 locus of the vaccine.These findings also support indirect antigen presentation as beingeffective in promoting antitumor activity and that allogeneic MHCmolecules enhance the effect.

Example 4 Establishment and Expansion of AD100-A1-B7.1 Cells

A human lung adenocarcinoma cell line (designated AD100) was establishedin 1994 at the University of Miami, derived from a patient with NSCLC.This cell line has been kept in culture in standard medium and is freeof contamination by Mycoplasma, virus, or other adventitious agents. Itis homogeneous, adherent to plastic, and grows at a rate of division ofapproximately 26 hours.

AD100 cells are transfected with plasmid cDNA, pBMG-Neo-B7.1 andpBMG-His-HLA A2 or with B45-Neo-CM-A1-B7.1. Transfected cells wereselected with G418 and Histidinol. Verification of correct sequences wasbased on restriction analysis and the expression of the relevant geneproducts, namely G418 or histidinol resistance for the vector sequence,HLA A1, A2, and B7.1 expression for the transfected cDNA. The minimumrequirement for their use as vaccine was the coexpression of HLA A1 orA2 plus B7.1 on at least 70% of the cells as shown in FIG. 1 a forrepresentative batches of vaccine cells.

AD100-A1-67.1 cells may be previously prepared and frozen in aliquots.The cryovial containing the cells is completely thawed rapidly using a37° C. water bath and gentle swirls. The cells are then transferred thecells immediately to a previously prepared sterile 15 ml conicalcentrifuge tube kept on ice. To this 15 ml conical centrifuge tube, 9 mlof Complete Media 1 (IMDM; FBS Certified heat inactivated-final con. 9%;Gentamicin-final conc. 0.04 mg/ml) is slowly added 1 to 2 drops at atime, while gently swirling the tube in order to uniformly mix cellswith media. This process should take 10 to 15 minutes. After all themedia is added, the cells are centrifuged cells at 300×g (1200 rpm) for10 minutes, at room temperature, with the brake set to “Low”. Thesupernatant is then gently aspirated away and the cells are resuspend in10 ml of Complete Media 2 (IMDM; FBS Certified heat inactivated-finalcon. 9%; Gentamicin-final conc. 0.04 mg/ml; Geneticin G-418-final conc.1 mg/ml), equilibrated to room temperature.

A cell count and viability test, using Trypan Blue @ 1:10 dilution isthen performed. Cells are then seeded at 2×10⁶ cells per T-175 tissueculture flask containing 35 ml of Complete Media 2. The seeded flasksare then incubated for 3 to 5 days in a 37° C. incubator with 5% CO₂.

Feeding Cells for Working Cell Bank

The cells should not be disturb until the third day of culture, when anassessment of whether cells have attached to the flask should be made.On the 3rd day of culture, a percentage of cells that have attachedneeds to be estimated. If ≧70% of the cells have attached to the flask,the media needs to be changed. Old media should be removed using anaspirating pipette and 50 ml of fresh Complete Media 1 pre-warmed to 37°C. should be added to each flask. The flasks are then returned to 37° C.incubator with 5% CO₂ for further culture. If, when observing cells onthe third day of culture, ≦70% of cells are deemed to be attached, theyneed to be left until the fifth day without changing media. After 3-5days in culture, remove flasks from the 37° C. incubator and determinepercentage of confluency. Cells need to be cultured until such time whenthey are deemed to be 90-95% confluent. The cells must be split when theconfluency reaches 90-95% per flask.

Harvesting the Cells with Trypsin EDTA for Working Cell Bank

After the cells reach 90-95% confluency, the cells are harvested byaspirating off the supernatant and by adding 12 ml of Trypsin-EDTApre-warmed to 37° C. to each flask. The cells are incubated at 37° C. inthis solution for approximately 20 minutes. After incubation, the flaskis vigorously shaken across its surface area, to ensure that the cellsare no longer adhering to the flask. 13 ml of Complete is then added toneutralize Trypsin-EDTA reaction. The supernatant containing the cellsthat have detached from the flask is then collected and transferred itto a sterile 50 ml or 250 ml conical centrifuge tube. Cell suspensionsfrom all the flasks should be combined and washed at the same time. Thecells are then centrifuged at 300×g (1200 rpm) for 10 minutes, at roomtemperature with the brake set to “Low”. The supernatant is thenaspirated off and the cells are resuspend in 15-30 ml of pre-warmed (to37° C.) Complete Media 2.

A cell count and viability test using Trypan Blue 1:10 dilution is themperformed.

New T-175 tissue culture flasks are then seeded at the density of2.0×10⁶ cells per flask, using pre-warmed (to 37° C.) Complete Media 2.The total volume of the Complete Media 2 in each T-175 tissue cultureflask should be 35 ml.

The above harvesting and expanding process is repeated approximatelyevery 7 days until 201 T-175 flasks can be seeded at one time. When thisthreshold is met, Complete Medium 2 is used to seed the cells for thefinal expansion. After the first 3-5 days of culture, when the cellshave attached and are ready to be fed, change to Complete Medium 1 isused. When the cells reach 90-95% confluency, the cells are harvested asabove. The cells are then washed twice in at least 200 ml of (4° C.)Wash Media (0.9% sodium chloride; 0.5% HAS; and 0.0067% USP sodiumbicarbonate). After the second wash, the cell pellet is resuspend inWash Media to the final volume of 200 ml. A cell count and viabilitytest using 1:70 dilution of Trypan Blue is again performed. The cellsare then irradiated at 12,000 rads using a Cobalt irradiator. The cellsare now ready for cryopreservation.

Cryopreservation of Expanded AD100-A1-B7.1 Cells

At least 80-120 cryovials should be labeled with cell identification,batch number, cell concentration, tech's initials, and date. The cellsare then centrifuged at 4° C., 300×g (1200 rpm), for 10 minutes, withthe brakes on. After which, the supernatant is aspirated off and thepelleted cells are placed on ice. The cells are then resuspend slowlywith gentle mixing, to a concentration of 200×10⁶/ml ice cold WashMedia. Ice cold Freezing Media (0.9% sodium chloride; 0.5% HAS; 0.0067%USP sodium bicarbonate; and 20% DMSO) is slowly added at a 1:1 ratio tohave a cell concentration of 100×10⁶/ml and DMSO concentration of 10%.The cells are then aliquoted at 0.5 ml (50×10⁶ cells) previouslyprepared cryovials on ice and then stored at −80° C. for 18-24 hours.After 24 hours, the frozen cells are transferred to the Liquid Nitrogenstorage tank.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains to thesame extent as if each was specifically and individually indicated to beincorporated by reference. The patents, published applications, andscientific literature referred to herein establish the knowledge ofthose with skill in the art and are hereby incorporated by reference intheir entirety to the same extent as if each was specifically andindividually indicated to be incorporated by reference. Any conflictbetween any reference cited herein and the specific teachings of thisspecification shall be resolved in favor of the latter. Likewise, anyconflict between an art-understood definition of a word or phrase and adefinition of the word or phrase as specifically taught in thisspecification shall be resolved in favor of the latter.

Example 5 1. Phase 1 Trial Design and Results

Three vaccinations, each of which was spaced 2 weeks apart, comprisedone course of treatment. At the end of the first course, patients whohad evidence of stable disease or responding NSCLC (by computedtomography scans), and no to moderate toxicity (grade≦2), were treatedwith a second course of vaccination. No patient was denied a second orthird course of treatment because of toxicity.

In the absence of tumor progression or severe toxicity (grade≧3), athird course of vaccination was given. No patients experienceddrug-related toxicity of grade≧3, and so all patients who did notprogress were eligible for the third course of vaccination.

Therefore, a total of three courses, or nine total vaccinations, werepossible in the study. Clinical and toxicity evaluations were donebefore and after each vaccination, and immunologic assessment was madebefore and after each course.

A. Survival Status and History of all Patients Tested in Trials.

The up-to-date survival curve is presented in FIG. 7. The full patienthistory and follow up is presented in Table 7.

B. Current Status of Responders.

Of six clinical responders, three have since died, the most recent inFebruary 2007 (patient # 14 below, and in Table 1). As of March 2007,there are three continuing survivors. The mean survival of the sixclinical responders is currently 59+ months (median=˜66+ (60 or 72+)months). The detailed status of the original six clinical responders isshown below (Patient # references to Table 7):

TABLE 7 Patient # Status Survival 4 Dead 23 mos. 16 Alive 48+ mos.   14Dead 60 mos. 10 Alive 72+ mos.   7 Dead 75 mos. 3 Alive 76+ mos.  

C. Up-to-Date Survival Curve (from Initiation of Trial to Present or toLast Survivor) Please see FIG. 7.

D. Percentage of the Patients Responded or Had an Adverse Effect.

As seen in Table 8, 19 patients were enrolled into the trial. It shouldbe noted that one patient was taken off study before receiving anyvaccinations, but he is still counted among the 19 patients.

Six of the 19 patients (32%) responded clinically with either partialresponse (PR) or stable disease (SD).

Three of the 19 patients (16%) experienced adverse events (grade 1 or 2)which were judged to be potentially vaccine-related. These adverseevents were comprised of: rash (1 patient), moderate arthritic pain inthe joints (1 patient), and chest pain (1 patient). Additionally, fourof the 19 patients (21%) developed some transient erythema at thevaccination site. The erythema is not considered to be an adverse eventsince it resolved within a week.

None of the 19 patients (0%) experienced drug-related serious adverseevents (SAEs). All SAEs were judged to be not vaccine-related. Four ofthe 19 patients (21%) experienced non-drug-related SAEs.

TABLE 8 HLA Location of Number of Clinical Additional Survival PatientSex Type Pathology Metastasis Vaccinations Response Chemotherapy (weeks)AE or SAE 1 F A2 A B 3 PD None 26 Rash 2 F A2 A L 3 PD Iressa 43 None 3F Non A P, L, A 9 SD None   328++ None 4 F Non A B 9 SD Gemzar; 100 None Carboplatin 5 M A1 B L 3 PD None 34 None 6 M Non A B, L 3 PD Iressa223  None Temodar 7 M Non A L 8, SD Revaccinated with 324  Pericardial 9PT 107; Effussion* Tarceva (1 yr. later) (recurrent) 8 F Non A L 3 PDGemzar 26 None 9 M A2 U L 3 PD None 12 None 10 F A2 A L, A 9 PR None  312++ Joint pain 11 M A2 S L 3 PD None 45 Chest pain 12 M A1 S A 8 PDNone 13 Renal Failure* 13 F A2 A B 4 PD None 11 Respiratory Failure* 14F A2 S L 9 SD Iressa; 238† None Tarceva 15 F A2 A L 3 PD Iressa 30 None16 F A1 B L 6 SD Velcade;   207++ None Alimta; Iressa 17 F A2 B Pl 3 PDGemzar; 23 None Navelbine 18 M Non B Li, L, T 3 PD None 49 None 19 F A2U M, Li 1 PD None  4 Pericardial Effussion* (tumor) Notes: Follow up asof Feb. 21, 2007. ++Still alive as of February 2007. †Died February2007. *Adverse event was determined to be not related to vaccine. Sex: F= female M = male HLA Type: A1 = HLA A1 A2 = HLA A2 Non = Non-HLA A1/A2Pathology: A = adenocarcinoma B = bronchial carcinoma S = squamous cellcarcinoma U - undifferentiated carcinoma Location of Metastasis: B =brain L = lung A = adrenal gland P = pancreas Pl = pleura Li = liver T =thoracic spinal column Clinical Response (responders shown in red): PD =progressive disease SD = stable disease PR = partial response

E. Patients Response to Different Levels of Vaccine.

Please see the graph in FIG. 8 for further detail. Patients who receiveda second or third course of vaccination fared much better in terms ofboth clinical response and survival. All five patients who received 8 or9 vaccinations were clinical responders. Of the clinical responders, 5of 6 (83%) received 8 or 9 vaccinations in their initial therapy. Of thenon-responders, 12 of 13 (92%) received 0-3 vaccinations.

F. Responder Breakdown Regarding Pathology Of NSCLC Cell Type(Adenocarcinoma, Bronchoalveolar, Squamous and Undifferentiated).

Of the 6 clinical responders, the pathology was as follows: 4 hadadenocarcinoma, 1 had bronchoalveolar carcinoma, and 1 had squamous cellcarcinoma. On a percentage basis, 4 of 11 (36%) patients withadenocarcinoma responded, 1 of 3 (33%) patients with bronchoalveolarcarcinoma responded, 1 of 3 (33%) patients with squamous cell carcinomaresponded, and 0 of 2 (0%) patients with undifferentiated carcinomaresponded. Please see Table 1 for further detail.

G. Comparison of Matched and Non-Matched HLA in Trials and Patients.

A multivariate analysis involving five covariates (HLA-matching, sex,race, pathology) showed no statistical significance of HLA-matching onoverall mortality.

Of the 19 patients, 13 were matched (3 at A1, 10 at A2), and 6 werenon-matched. Of the 6 clinical responders, 3 were HLA matched, and 3were non-matched. Among matched patients, 1 of the 3 (33%) A1-matchedpatients were clinical responders, and 2 of 10 (20%) A2-matched patientswere clinical responders. Among non-matched patients, 3 of 6 (50%) wereclinical responders.

It should be noted that logistic regression analyses of age, sex, race,pathology, and HLA-matching of vaccine showed that none of these factorswere statistically significantly related (P≧0.10 in all instances) toclinical response.

Univariate proportional hazards regression analysis suggested a possiblyhigher mortality rate in patients receiving HLA-matched vaccine (hazardratio=4.5; 90% CI, 1.1 to 17.2), and a possibly lower mortality rate inpatients with adenocarcinoma (hazard ratio=0.3; 90% CI, 0.1 to 1.0). Amultivariate analysis involving five covariates (HLA-matching, age, sex,race, pathology) however, discounted an adverse effect of HLA-matchingof vaccine on overall mortality; the corresponding adjusted hazard ratiowas 1.9 (P=0.51). The adjusted hazard ratio for adenocarcinoma versusother pathologies was 0.2 (P=0.11), which is within the realm of chanceat conventional levels of significance.

BPV-1-B7.1-HLA A1 vector sequence XbaT SEQ ID NO:1     ~~~~~~     1TCTAGAGAGC TTGGCCCATT GCATACGTTG TATCCATATC ATAATATGTA AGATCTCTCGAACCGGGTAA CGTATGCAAC ATAGGTATAG TATTATACAT    51 CATTTATATT GGCTCATGTCCAACATTACC GCCATGTTGA CATTGATTAT GTAAATATAA CCGAGTACAG GTTGTAATGGCGGTACAACT GTAACTAATA   101 TGACTAGTTA TTAATAGTAA TCAATTACGG GGTCATTAGTTCATAGCCCA ACTGATCAAT AATTATCATT AGTTAATGCC CCAGTAATCA AGTATCGGGT   151TATATGGAGT TCCGCGTTAC ATAACTTACG GTAAATGGCC CGCCTGGCTG ATATACCTCAAGGCGCAATG TATTGAATGC CATTTACCGG GCGGACCGAC   201 ACCGCCCAAC GACCCCCGCCCATTGACGTC AATAATGACG TATGTTCCCA TGGCGGGTTG CTGGGGGCGG GTAACTGCAGTTATTACTGC ATACAAGGGT   251 TAGTAACGCC AATAGGGACT TTCCATTGAC GTCAATGGGTGGAGTATTTA ATCATTGCGG TTATCCCTGA AAGGTAACTG CAGTTACCCA CCTCATAAAT   301CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATCATA TGCCAAGTAC GCCATTTGACGGGTGAACCG TCATGTAGTT CACATAGTAT ACGGTTCATG   351 GCCCCCTATT GACGTCAATGACGGTAAATG GCCCGCCTGG CATTATGCCC CGGGGGATAA CTGCAGTTAC TGCCATTTACCGGGCGGACC GTAATACGGG   401 ACTACATGAC CTTATGGGAC TTTCCTACTT GGCAGTACATCTACGTATTA TCATGTACTG GAATACCCTG AAAGGATGAA CCGTCATGTA GATGCATAAT   451GTCATCGCTA TTACCATGGT GATGCGGTTT TGGCAGTACA TCAATGGGCG CAGTAGCGATAATGGTACCA CTACGCCAAA ACCGTCATGT AGTTACCCGC   501 TGGATAGCGG TTTGACTCACGGGGATTTCC AAGTCTCCAC CCCATTGACG ACCTATCGCC AAACTGAGTG CCCCTAAAGGTTCAGAGGTG GGGTAACTGC   551 TCAATGGGAG TTTGTTTTGG CACCAAAATC AACGGGACTTTCCAAAATGT AGTTACCCTC AAACAAAACC GTGGTTTTAG TTGCCCTGAA AGGTTTTACA   601CGTAACAACT CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GCATTGTTGAGGCGGGGTAA CTGCGTTTAC CCGCCATCCG CACATGCCAC   651 GGAGGTCTAT ATAAGCAGAGCTCGTTTAGT GAACCGTCAG ATCGCCTGGA CCTCCAGATA TATTCGTCTC GAGCAAATCACTTGGCAGTC TAGCGGACCT   701 GACGCCATCC ACGCTGTTTT GACCTCCATA GAAGACACCGGGACCGATCC CTGCGGTAGG TGCGACAAAA CTGGAGGTAT CTTCTGTGGC CCTGGCTAGG   751AGCCTCCGGT CGATCGACCG ATCCTGAGAA CTTCAGGGTG AGTTTGGGGA TCGGAGGCCAGCTAGCTGGC TAGGACTCTT GAAGTCCCAC TCAAACCCCT   801 CCCTTGATTG TTCTTTCTTTTTCGCTATTG TAAAATTCAT GTTATATGGA GGGAACTAAC AAGAAAGAAA AAGCGATAACATTTTAAGTA CAATATACCT   851 GGGGGCAAAG TTTTCAGGGT GTTGTTTAGA ATGGGAAGATGTCCCTTGTA CCCCCGTTTC AAAAGTCCCA CAACAAATCT TACCCTTCTA CAGGGAACAT   901TCACCATGGA CCCTCATGAT AATTTTGTTT CTTTCACTTT CTACTCTGTT AGTGGTACCTGGGAGTACTA TTAAAACAAA GAAAGTGAAA GATGAGACAA   951 GACAACCATT GTCTCCTCTTATTTTCTTTT CATTTTCTGT AACTTTTTCG CTGTTGGTAA CAGAGGAGAA TAAAAGAAAAGTAAAAGACA TTGAAAAAGC  1001 TTAAACTTTA GCTTGCATTT GTAACGAATT TTTAAATTCACTTTTGTTTA AATTTGAAAT CGAACGTAAA CATTGCTTAA AAATTTAAGT GAAAACAAAT  1051TTTGTCAGAT TGTAAGTACT TTCTCTAATC ACTTTTTTTT CAAGGCAATC AAACAGTCTAACATTCATGA AAGAGATTAG TGAAAAAAAA GTTCCGTTAG  1101 AGGGTATATT ATATTGTACTTCAGCACAGT TTTAGAGAAC AATTGTTATA TCCCATATAA TATAACATGA AGTCGTGTCAAAATCTCTTG TTAACAATAT  1151 ATTAAATGAT AAGGTAGAAT ATTTCTGCAT ATAAATTCTGGCTGGCGTGG TAATTTACTA TTCCATCTTA TAAAGACGTA TATTTAAGAC CGACCGCACC  1201AAATATTCTT ATTGGTAGAA ACAACTACAC CCTGGTCATC ATCCTGCCTT TTTATAAGAATAACCATCTT TGTTGATGTG GGACCAGTAG TAGGACGGAA  1251 TCTCTTTATG GTTACAATGATATACACTGT TTGAGATGAG GATAAAATAC AGAGAAATAC CAATGTTACT ATATGTGACAAACTCTACTC CTATTTTATG  1301 TCTGAGTCCA AACCGGGCCC CTCTGCTAAC CATGTTCATGCCTTCTTCTC AGACTCAGGT TTGGCCCGGG GAGACGATTG GTACAAGTAC GGAAGAAGAG  1351TTTCCTACAG CTCCTGGGCA ACGTGCTGGT TGTTGTGCTG TCTCATCATT AAAGGATGTCGAGGACCCGT TGCACGACCA ACAACACGAC AGAGTAGTAA                XhoI        Start B7.1 (CD80)          SEQ ID NO:2       MetGly HisThrArg ArgGlnGly               ~~~~~~~      ~~~~~~~~~~~~~~~~~~~~~~~~~~  1401 TTGGCAAAGAATTCCTCGAG GAAGCCATGG GCCACACACG GAGGCAGGGA AACCGTTTCT TAAGGAGCTCCTTCGGTACC CGGTGTGTGC CTCCGTCCCT ThrSerProSer LysCysPro TyrLeuAsnPhePheGlnLeu LeuValLeu~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1451 ACATCACCATCCAAGTGTCC ATACCTCAAT TTCTTTCAGC TCTTGGTGCTG TGTAGTGGTA GGTTCACAGGTATGGAGTTA AAGAAAGTCG AGAACCACGA AlaGlyLeu SerHisPheCys SerGlyValIleHisVal ThrLysGluVal~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1501 GCTGGTCTTTCTCACTTCT GTTCAGGTGT TATCCACGTG ACCAAGGAAG CCGACCAGAA AGAGTGAAGACAAGTCCACA ATAGGTGCAC TGGTTCCTTC *VLysGluVal AlaThrLeu SerCysGlyHisAsnValSer ValGluGlu~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1551 TGAAAGAAGTGGCAACGCTG TCCTGTGGTC ACAATGTTTC TGTTGAAGAG ACTTTCTTCA CCGTTGCGACAGGACACCAG TGTTACAAAG ACAACTTCTC LeuAlaGlnThr ArgIleTyr TrpGlnLysGluLysLysMet ValLeuThr*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1601 CTGGCACAAACTCGCATCTA CTGGCAAAAG GAGAAGAAAA TGGTGCTGAC GACCGTGTTT GAGCGTAGATGACCGTTTTC CTCTTCTTTT ACCACGACTG *MetMetSer GlyAspMetAsn IleTrpProGluTyrLys AsnArgThrIle*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1651 TATGATGTCTGGGGACATGA ATATATGGCC CGAGTACAAG AACCGGACCA ATACTACAGA CCCCTGTACTTATATACCGG GCTCATGTTC TTGGCCTGGT *IPheAspIle ThrAsnAsn LeuSerIleValIleLeuAla LeuArgPro~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1701 TCTTTGATATCACTAATAAC CTCTCCATTG TGATCCTGGC TCTGCGCCCA AGAAACTATA GTGATTATTGGAGAGGTAAC ACTAGGACCG AGACGCGGGT SerAspGluGly ThrTyrGlu CysValValLeuLysTyrGlu LysAspAla*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1751 TCTGACGAGGGCACATACGA GTGTGTTGTT CTGAAGTATG AAAAAGACGC AGACTGCTCC CGTGTATGCTCACACAACAA GACTTCATAC TTTTTCTGCG *PheLysArg GluHisLeuAla GluValThrLeuSerVal LysAlaAspPhe*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1801 TTTCAAGCGGGAACACCTGG CTGAAGTGAC GTTATCAGTC AAAGCTGACT AAAGTTCGCC CTTGTGGACCGACTTCACTG CAATAGTCAG TTTCGACTGA *PProThrPro SerIleSer AspPheGluIleProThrSer AsnIleArg~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1851 TCCCTACACCTAGTATATCT GACTTTGAAA TTCCAACTTC TAATATTAGA AGGGATGTGG ATCATATAGACTGAAACTTT AAGGTTGAAG ATTATAATCT ArgIleIleCys SerThrSer GlyGlyPheProGluProHis LeuSerTrp*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1901 AGGATAATTTGCTCAACCTC TGGAGGTTTT CCAGAGCCTC ACCTCTCCTG TCCTATTAAA CGAGTTGGAGACCTCCAAAA GGTCTCGGAG TGGAGAGGAC *LeuGluAsn GlyGluGluLeu AsnAlaIleAsnThrThr ValSerGlnAsp*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  1951 GTTGGAAAATGGAGAAGAAT TAAATGCCAT CAACACAACA GTTTCCCAAG CAACCTTTTA CCTCTTCTTAATTTACGGTA GTTGTGTTGT CAAAGGGTTC *AProGluThr GluLeuTyr AlaValSerSerLysLeuAsp PheAsnMet~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  2001 ATCCTGAAACTGAGCTCTAT GCTGTTAGCA GCAAACTGGA CTTCAATATG TAGGACTTTG ACTCGAGATACGACAATCGT CGTTTGACCT GAAGTTATAC ThrThrAsnHis SerPheMet CysLeuIleLysTyrGlyHis LeuArgVal*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  2051 ACAACCAACCACAGCTTCAT GTGTCTCATC AAGTATGGAC ATTTAAGAGT TGTTGGTTGG TGTCGAAGTACACAGACTAG TTCATACCTG TAAATTCTCA *AsnGlnThr PheAsnTrpAsn ThrThrLysGlnGluHis PheProAspAsn*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  2101 GAATCAGACCTTCAACTGGA ATACAACCAA GCAAGAGCAT TTTCCTGATA CTTAGTCTGG AAGTTGACCTTATGTTGGTT CGTTCTCGTA AAAGGACTAT *ALeuLeuPro SerTrpAla IleThrLeuIleSerValAsn GlyIlePhe~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  2151 ACCTGCTCCCATCCTGGGCC ATTACCTTAA TCTCAGTAAA TGGAATTTTT TGGACGAGGG TAGGACCCGGTAATGGAATT AGAGTCATTT ACCTTAAAAA ValIleCysCys LeuThrTyr CysPheAlaProArgCysArg GluArgArg*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  2201 GTGATATGCTGCCTGACCTA CTGCTTTGCC CCAAGATGCA GAGAGAGAAG CACTATACGA CGGACTGGATGACGAAACGG GGTTCTACGT CTCTCTCTTC *ArgAsnGlu ArgLeuArgArg GluSerValArgProVal *** ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Stop  2251GAGGAATGAG AGATTGAGAA GGGAAAGTGT ACGCCCTGTA TAACAGACTA CTCCTTACTCTCTAACTCTT CCCTTTCACA TGCGGGACAT ATTGTCTGAT                                                  XhoI                                                  ~~~~  2301 GTCAAATTAAGCCGAATTCT GCAGATATCC ATCACACTGG CGGCCGCTCG CAGTTTAATT CGGCTTAAGACGTCTATAGG TAGTGTGACC GCCGGCGAGC XhoI ~~  2351 AGGAATTCAC TCCTCAGGTGCAGGCTGCCT ATCAGAAGGT GGTGGCTGGT TCCTTAAGTG AGGAGTCCAC GTCCGACGGATAGTCTTCCA CCACCGACCA  2401 GTGGCCAATG CCCTGGCTCA CAAATACCAC TGAGATCTTTTTCCCTCTGC CACCGGTTAC GGGACCGAGT GTTTATGGTG ACTCTAGAAA AAGGGAGACG  2451CAAAAATTAT GGGGACATCA TGAAGCCCCT TGAGCATCTG ACTTCTGGCT GTTTTTAATACCCCTGTAGT ACTTCGGGGA ACTCGTAGAC TGAAGACCGA  2501 AATAAAGGAA ATTTATTTTCATTGCAATAG TGTGTTGGAA TTTTTTGTGT TTATTTCCTT TAAATAAAAG TAACGTTATCACACAACCTT AAAAAACACA  2551 CTCTCACTCG GAAGGACATA TGGGAGGGCA AATCATTTAAAACATCAGAA GAGAGTGAGC CTTCCTGTAT ACCCTCCCGT TTAGTAAATT TTGTAGTCTT  2601TGAGTATTTG GTTTAGAGTT TGGCAACATA TGCCCATATG CTGGCTGCCA ACTCATAAACCAAATCTCAA ACCGTTGTAT ACGGGTATAC GACCGACGGT  2651 TGAACAAAGG TTGGCTATAAAGAGGTCATC AGTATATGAA ACAGCCCCCT ACTTGTTTCC AACCGATATT TCTCCAGTAGTCATATACTT TGTCGGGGGA  2701 GCTGTCCATT CCTTATTCCA TAGAAAAGCC TTGACTTGAGGTTAGATTTT CGACAGGTAA GGAATAAGGT ATCTTTTCGG AACTGAACTC CAATCTAAAA  2751TTTTATATTT TGTTTTGTGT TATTTTTTTC TTTAACATCC CTAAAATTTT AAAATATAAAACAAAACACA ATAAAAAAAG AAATTGTAGG GATTTTAAAA  2801 CCTTACATGT TTTACTAGCCAGATTTTTCC TCCTCTCCTG ACTACTCCCA GGAATGTACA AAATGATCGG TCTAAAAAGGAGGAGAGGAC TGATGAGGGT                                           BamHI                                          ~~~~~~~  2851 GTCATAGCTGTCCCTCTTCT CTTATGGAGA TCCCTCGACG GATCCCTAGA CAGTATCGAC AGGGAGAAGAGAATACCTCT AGGGAGCTGC CTAGGGATCT  2901 GTCGAGGCGA TGCGGCGCAG CACCATGGCCTGAAATAACC TCTGAAAGAG CAGCTCCGCT ACGCCGCGTC GTGGTACCGG ACTTTATTGGAGACTTTCTC  2951 GAACTTGGTT AGGTACCTTG GTTTTTAAAA CCAGCCTGGA GTAGAGCAGACTTGAACCAA TCCATGGAAC CAAAAATTTT GGTCGGACCT CATCTCGTCT  3001 TGGGTTAAGGTGAGTGACCC CTCAGCCCTG GACATTCTTA GATGAGCCCC ACCCAATTCC ACTCACTGGGGAGTCGGGAC CTGTAAGAAT CTACTCGGGG  3051 CTCAGGAGTA GAGAATAATG TTGAGATGAGTTCTGTTGGC TAAAATAATC GAGTCCTCAT CTCTTATTAC AACTCTACTC AAGACAACCGATTTTATTAG  3101 AAGGCTAGTC TTTATAAAAC TGTCTCCTCT TCTCCTAGCT TCGATCCAGATTCCGATCAG AAATATTTTG ACAGAGGAGA AGAGGATCGA AGCTAGGTCT  3151 GAGAGACCTGGGCGGAGCTG GTCGCTGCTC AGGAACTCCA GGAAAGGAGA CTCTCTGGAC CCGCCTCGACCACCGACGAG TCCTTGAGGT CCTTTCCTCT  3201 AGCTGAGGTT ACCACGCTGC GAATGGGTTTACGGAGATAG CTGGCTTTCC TCGACTCCAA TGGTGCGACG CTTACCCAAA TGCCTCTATCGACCGAAAGG  3251 GGGGTGAGTT CTCGTAAACT CCAGAGCAGC GATAGGCCGT AATATCGGGGCCCCACTCAA GAGCATTTGA GGTCTCGTCG CTATCCGGCA TTATAGCCCC  3301 AAAGCACTATAGGGACATGA TGTTCCACAC GTCACATGGG TCGTCCTATC TTTCGTGATA TCCCTGTACTACAAGGTGTG CAGTGTACCC ACCAGGATAG  3351 CGAGCCAGTC GTGCCAAAGG GGCGGTCCCGCTGTGCACAC TGGCGCTCCA GCTCGGTCAG CACGGTTTCC CCGCCAGGGC GACACGTGTGACCGCGAGGT  3401 GGGAGCTCTG CACTCCGCCC GAAAAGTGCG CTCGGCTCTG CCAGGACGCGCCCTCGAGAC GTGAGGCGGG CTTTTCACGC GAGCCGAGAC GGTCCTGCGC  3451 GGGCGCGTGACTATGCGTGG GCTGGAGCAA CCGCCTGCTG GGTGCAAACC CCCGCGCACT GATACGCACCCGACCTCGTT GGCGGACGAC CCACGTTTGG  3501 CTTTGCGCCC GGACTCGTCC AACGACTATAAAGAGGGCAG GCTGTCCTCT GAAACGCGGG CCTGAGCAGG TTGCTGATAT TTCTCCCGTCCGACAGGAGA  3551 AAGCGTCACC ACGACTTCAA CGTCCTGAGT ACCTTCTCCT CACTTACTCCTTCGCAGTGG TGCTGAAGTT GCAGGACTCA TGGAAGAGGA GTGAATGAGG                                    SalI                                   ~~~~~~  3601 GTAGCTCCAG CTTCACCACCAAGCTCCTCG ACGTCGACCC CAGACGCCGA CATCGAGGTC GAAGTGGTGG TTCGAGGAGCTGCAGCTGGG GTCTGCGGCT Start SEQ ID NO: 3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ HLA A1    MetAlaValMetAlaPro ArgThrLeuLeu LeuLeuLeu SerGlyAla  3651 GGATGGCCGT CATGGCGCCCCGAACCCTCC TCCTGCTACT CTCGGGGGCC CCTACCGGCA GTACCGCGGG GCTTGGGAGGAGGACGATGA GAGCCCCCGG~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ LeuAlaLeuThrGlnThrTrp AlaGlySer HisSerMetArg TyrPhePhe*  3701 CTGGCCCTGA CCCAGACCTGGGCGGGCTCC CACTCCATGA GGTATTTCTT GACCGGGACT GGGTCTGGAC CCGCCCGAGGGTGAGGTACT CCATAAAGAA~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *ThrSerValSerArgProGly ArgGlyGlu ProArgPhe IleAlaValGly*  3751 CACATCCGTGTCCCGGCCCG GCCGCGGGGA GCCCCGCTTC ATCGCCGTGG GTGTAGGCAC AGGGCCGGGCCGGCGCCCCT CGGGGCGAAG TAGCGGCACC~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *GTyrValAspAspThrGln PheValArgPhe AspSerAsp AlaAlaSer  3801 GCTACGTGGA CGACACGCAGTTCGTGCGGT TCGACAGCGA CGCCGCGAGC CGATGCACCT GCTGTGCGTC AAGCACGCCAAGCTGTCGCT GCGGCGCTCG~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ GlnLysMetGluProArgAla ProTrpIle GluGlnGluGly ProGluTyr*  3851 CAGAAGATGG AGCCGCGGGCGCCGTGGATA GAGCAGGAGG GGCCGGAGTA GTCTTCTACC TCGGCGCCCG CGGCACCTATCTCGTCCTCC CCGGCCTCAT~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *TrpAspGlnGluThrArgAsn MetLysAla HisSerGln ThrAspArgAla*  3901 TTGGGACCAGGAGACACGGA ATATGAAGGC CCACTCACAG ACTGACCGAG AACCCTGGTC CTCTGTGCCTTATACTTCCG GGTGAGTGTC TGACTGGCTC~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *AAsnLeuGlyThrLeuArg GlyTyrTyrAsn GlnSerGlu AspGlySer  3951 CGAACCTGGG GACCCTGCGCGGCTACTACA ACCAGAGCGA GGACGGTTCT GCTTGGACCC CTGGGACGCG CCGATGATGTTGGTCTCGCT CCTGCCAAGA~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ HisThrIleGlnIleMetTyr GlyCysAsp ValGlyProAsp GlyArgPhe  4001 CACACCATCC AGATAATGTATGGCTGCGAC GTGGGGCCGG ACGGGCGCTT GTGTGGTAGG TCTATTACAT ACCGACGCTGCACCCCGGCC TGCCCGCGAA~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *LeuArgGlyTyrArgGlnAsp AlaTyrAsp GlyLysAsp TyrIleAlaLeu*  4051 CCTCCGCGGGTACCGGCAGG ACGCCTACGA CGGCAAGGAT TACATCGCCC GGAGGCGCCC ATGGCCGTCCTGCGGATGCT GCCGTTCCTA ATGTAGCGGG~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *LAsnGluAspLeuArgSer TrpThrAlaAla AspMetAla AlaGlnIle  4101 TGAACGAGGA CCTGCGCTCTTGGACCGCGG CGGACATGGC GGCTCAGATC ACTTGCTCCT GGACGCGAGA ACCTGGCGCCGCCTGTACCG CCGAGTCTAG~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ThrLysArgLysTrpGluAla AlaHisAla AlaGluGlnArg ArgValTyr*  4151 ACCAAGCGCA AGTGGGAGGCGGCCCATGCG GCGGAGCAGC GGAGAGTCTA TGGTTCGCGT TCACCCTCCG CCGGGTACGCCGCCTCGTCG CCTCTCAGAT~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *LeuAspGlyArgCysValAsp GlyLeuArg ArgTyrLeu GluAsnGlyLys*  4201 CCTGGATGGCCGGTGCGTGG ACGGGCTCCG CAGATACCTG GAGAACGGGA GGACCTACCG GCCACGCACCTGCCCGAGGC GTCTATGGAC CTCTTGCCCT~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *LGluThrLeuGlnArgThr AspProProLys ThrHisMet ThrHisHis  4251 AGGAGACGCT GCAGCGCACGGACCCCCCCA AGACACATAT GACCCACCAC TCCTCTGCGA CGTCGCGTGC CTGGGGGGGTTCTGTGTATA CTGGGTGGTG~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ProIleSerAspHisGluAla ThrLeuArg CysTrpAlaLeu GlyPheTyr*  4301 CCCATCTCTG ACCATGAGGCCACCCTGAGG TGCTGGGCCC TGGGCTTCTA GGGTAGAGAC TGGTACTCCG GTGGGACTCCACGACCCGGG ACCCGAAGAT~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *ProAlaGluIleThrLeuThr TrpGlnArg AspGlyGlu AspGlnThrGln*  4351 CCCTGCGGAGATCACACTGA CCTGGCAGCG GGATGGGGAG GACCAGACCC GGGACGCCTC TAGTGTGACTGGACCGTCGC CCTACCCCTC CTGGTCTGGG~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *GAspThrGluLeuValGlu ThrArgProAla GlyAspGly ThrPheGln  4401 AGGACACGGA GCTCGTGGAGACCAGGCCTG CAGGGGATGG AACCTTCCAG TCCTGTGCCT CGAGCACCTC TGGTCCGGACGTCCCCTACC TTGGAAGGTC~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ LysTrpAlaAlaValValVal ProSerGly GluGluGlnArg TyrThrCys*  4451 AAGTGGGCGG CTGTGGTGGTGCCTTCTGGA GAGGAGCAGA GATACACCTG TTCACCCGCC GACACCACCA CGGAAGACCTCTCCTCGTCT CTATGTGGAC *HisValGln HisGluGlyLeu ProLysPro LeuThrLeuArgTrpGluLeu* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 4501 CCATGTGCAG CATGAGGGTC TGCCCAAGCC CCTCACCCTG AGATGGGAGC GGTACACGTCGTACTCCCAG ACGGGTTCGG GGAGTGGGAC TCTACCCTCG *LSerSerGln ProThrIleProIleValGly IleIleAla GlyLeuVal~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  4551 TGTCTTCCCAGCCCACCATC CCCATCGTGG GCATCATTGC TGGCCTGGTT ACAGAAGGGT CGGGTGGTAGGGGTAGCACC CGTAGTAACG ACCGGACCAA LeuLeuGlyAla ValIleThr GlyAlaValValAlaAlaVal MetTrpArg*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  4601 CTCCTTGGAGCTGTGATCAC TGGAGCTGTG GTCGCTGCCG TGATGTGGAG GAGGAACCTC GACACTAGTGACCTCGACAC CAGCGACGGC ACTACACCTC *ArgLysSer SerAspArgLys GlyGlySerTyrThrGln AlaAlaSerSer*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  4651 GAGGAAGAGCTCAGATAGAA AAGGAGGGAG TTACACTCAG GCTGCAAGCA CTCCTTCTCG AGTCTATCTTTTCCTCCCTC AATGTGAGTC CGACGTTCGT *SAspSerAla GlnGlySer AspValSerLeuThrAlaCys LysVal***~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Stop  4701 GTGACAGTGCCCAGGGCTCT GATGTGTCCC TCACAGCTTG TAAAGTGTGA CACTGTCACG GGTCCCGAGACTACACAGGG AGTGTCGAAC ATTTCACACT  4751 GACAGCTGCC TTGTGTGGGA CTGAGAGGCAAGAGTTGTTC CTGCCCTTCC CTGTCGACGG AACACACCCT GACTCTCCGT TCTCAACAAGGACGGGAAGG  4801 CTTTGTGACT TGAAGAACCC TGACTTTGTT TCTGCAAAGG CACCTGCATGGAAACACTGA ACTTCTTGGG ACTGAAACAA AGACGTTTCC GTGGACGTAC  4851 TGTCTGTGTTCGTGTAGGCA TAATGTGAGG AGGTGGGGAG AGCACCCCAC ACAGACACAA GCACATCCGTATTACACTCC TCCACCCCTC TCGTGGGGTG  4901 CCCCTGTCCA CCATGACCCT CTTCCCACGCTGACCTGTGC TCCCTCCCCA GGGGACAGGT GGTACTGGGA GAAGGGTGCG ACTGGACACGAGGGAGGGGT  HindIII                          BamHI             SalI ~~~~~~                           ~~~~~~            ~~  4951 AAAGCTTGATATCCAATTCC TGCAGCCCGG GGGATCCACT TTTTCTAGGT TTTCGAACTA TAGGTTAAGGACGTCGGGCC CCCTAGGTGA AAAAGATCCA SalI ~~~~  5001 CGACCGATCC TGAGAACTTCAGGGTGAGTT TGGGGACCCT TGATTGTTCT GCTGGCTAGG ACTCTTGAAG TCCCACTCAAACCCCTGGGA ACTAACAAGA  5051 TTCTTTTTCG CTATTGTAAA ATTCATGTTA TATGGAGGGGGCAAAGTTTT AAGAAAAAGC GATAACATTT TAAGTACAAT ATACCTCCCC CGTTTCAAAA  5101CAGGGTGTTG TTTAGAATGG GAAGATGTCC CTTGTATCAC CATGGACCCT GTCCCACAACAAATCTTACC CTTCTACAGG GAACATAGTG GTACCTGGGA  5151 CATGATAATT TTGTTTCTTTCACTTTCTAC TCTGTTGACA ACCATTGTCT GTACTATTAA AACAAAGAAA GTGAAAGATGAGACAACTGT TGGTAACAGA  5201 CCTCTTATTT TCTTTTCATT TTCTGTAACT TTTTCGTTAAACTTTAGCTT GGAGAATAAA AGAAAAGTAA AAGACATTGA AAAAGCAATT TGAAATCGAA  5251GCATTTGTAA CGAATTTTTA AATTCACTTT TGTTTATTTG TCAGATTGTA CGTAAACATTGCTTAAAAAT TTAAGTGAAA ACAAATAAAC AGTCTAACAT  5301 AGTACTTTCT CTAATCACTTTTTTTTCAAG GCAATCAGGG TATATTATAT TCATGAAAGA GATTAGTGAA AAAAAAGTTCCGTTAGTCCC ATATAATATA  5351 TGTACTTCAG CACAGTTTTA GAGAACAATT GTTATAATTAAATGATAAGG ACATGAAGTC GTGTCAAAAT CTCTTGTTAA CAATATTAAT TTACTATTCC  5401TAGAATATTT CTGCATATAA ATTCTGGCTG GCGTGGAAAT ATTCTTATTG ATCTTATAAAGACGTATATT TAAGACCGAC CGCACCTTTA TAAGAATAAC  5451 GTAGAAACAA CTACACCCTGGTCATCATCC TGCCTTTCTC TTTATGGTTA CATCTTTGTT GATGTGGGAC CAGTAGTAGGACGGAAAGAG AAATACCAAT  5501 CAATGATATA CACTGTTTGA GATGAGGATA AAATACTCTGAGTCCAAACC GTTACTATAT GTGACAAACT CTACTCCTAT TTTATGAGAC TCAGGTTTGG  5551GGGCCCCTCT GCTAACCATG TTCATGCCTT CTTCTCTTTC CTACAGCTCC CCCGGGGAGACGATTGGTAC AAGTACGGAA GAAGAGAAAG GATGTCGAGG  5601 TGGGCAACGT GCTGGTTGTTGTGCTGTCTC ATCATTTTGG CAAAGAATTC ACCCGTTGCA CGACCAACAA CACGACAGAGTAGTAAAACC GTTTCTTAAG  5651 CTCGACCAGT GCAGGCTGCC TATCAGAAAG TGGTGGCTGGTGTGGCTAAT GAGCTGGTCA CGTCCGACGG ATAGTCTTTC ACCACCGACC ACACCGATTA  5701GCCCTGGCCC ACAAGTATCA CTAAGCTCGC TTTCTTGCTG TCCAATTTCT CGGGACCGGGTGTTCATAGT GATTCGAGCG AAAGAACGAC AGGTTAAAGA  5751 ATTAAAGGTT CCTTTGTTCCCTAAGTCCAA CTACTAAACT GGGGGATATT TAATTTCCAA GGAAACAAGG GATTCAGGTTGATGATTTGA CCCCCTATAA  5801 ATGAAGGGCC TTGAGCATCT GGATTCTGCC TAATAAAAAACATTTATTTT TACTTCCCGG AACTCGTAGA CCTAAGACGG ATTATTTTTT GTAAATAAAA  5851CATTGCAATG ATGTATTTAA ATTATTTCTG AATATTTTAC TAAAAAGGGA GTAACGTTACTACATAAATT TAATAAAGAC TTATAAAATG ATTTTTCCCT  5901 ATGTGGGAGG TCAGTGCATTTAAAACATAA AGAAATGAAG AGCTAGTTCA TACACCCTCC AGTCACGTAA ATTTTGTATTTCTTTACTTC TCGATCAAGT  5951 AACCTTGGGA AAATACACTA TATCTTAAAC TCCATGAAAGAAGGTGAGGC TTGGAACCCT TTTATGTGAT ATAGAATTTG AGGTACTTTC TTCCACTCCG  6001TGCAAACAGC TAATGCACAT TGGCAACAGC CCCTGATGCC TATGCCTTAT ACGTTTGTCGATTACGTGTA ACCGTTGTCG GGGACTACGG ATACGGAATA  6051 TCATCCCTCA GAAAAGGATTCAAGTAGAGG CTTGATTTGG AGGTTAAAGT AGTAGGGAGT CTTTTCCTAA GTTCATCTCCGAACTAAACC TCCAATTTCA  6101 TTTGCTATGC TGTATTTTAC ATTACTTATT GTTTTAGCTGTCCTCATGAA AAACGATACG ACATAAAATG TAATGAATAA CAAAATCGAC AGGAGTACTT  6151TGTCTTTTCA CTACCCATTT GCTTATCCTG CATCTCTCAG CCTTGACTCC ACAGAAAAGTGATGGGTAAA CGAATAGGAC GTAGAGAGTC GGAACTGAGG  6201 ACTCAGTTCT CTTGCTTAGAGATACCACCT TTCCCCTGAA GTGTTCCTTC TGAGTCAAGA GAACGAATCT CTATGGTGGAAAGGGGACTT CACAAGGAAG  6251 CATGTTTTAC GGCGAGATGG TTTCTCCTCG CCTGGCCACTCAGCCTTAGT GTACAAAATG CCGCTCTACC AAAGAGGAGC GGACCGGTGA GTCGGAATCA  6301TGTCTCTGTT GTCTTATAGA GGTCTACTTG AAGAAGGAAA AACAGGGGGC ACAGAGACAACAGAATATCT CCAGATGAAC TTCTTCCTTT TTGTCCCCCG  6351 ATGGTTTGAC TGTCCTGTGAGCCCTTCTTC CCTGCCTCCC CCACTCACAG TACCAAACTG ACAGGACACT CGGGAAGAAGGGACGGAGGG GGTGAGTGTC  6401 TGACCCGGAA TCTGCAGTGC TAGTCTCCCG GAACTATCACTCTTTCACAG ACTGGGCCTT AGACGTCACG ATCAGAGGGC CTTGATAGTG AGAAAGTGTC  6451TCTGCTTTGG AAGGACTGGG CTTAGTATGA AAAGTTAGGA CTGAGAAGAA AGACGAAACCTTCCTGACCC GAATCATACT TTTCAATCCT GACTCTTCTT  6501 TTTGAAAGGG GGCTTTTTGTAGCTTGATAT TCACTACTGT CTTATTACCC AAACTTTCCC CCGAAAAACA TCGAACTATAAGTGATGACA GAATAATGGG  6551 TATCATAGGC CCACCCCAAA TGGAAGTCCC ATTCTTCCTCAGGATGTTTA ATAGTATCCG GGTGGGGTTT ACCTTCAGGG TAAGAAGGAG TCCTACAAAT  6601AGATTAGCAT TCAGGAAGAG ATCAGAGGTC TGCTGGCTCC CTTATCATGT TCTAATCGTAAGTCCTTCTC TAGTCTCCAG ACGACCGAGG GAATAGTACA  6651 CCCTTATGGT GCTTCTGGCTCTGCAGTTAT TAGCATAGTG TTACCATCAA GGGAATACCA CGAAGACCGA GACGTCAATAATCGTATCAC AATGGTAGTT  6701 CCACCTTAAC TTCATTTTTC TTATTCAATA CCTAGGTAGGTAGATGCTAG GGTGGAATTG AAGTAAAAAG AATAAGTTAT GGATCCATCC ATCTACGATC  6751ATTCTGGAAA TAAAATATGA GTCTCAAGTG GTCCTTGTCC TCTCTCCCAG TAAGACCTTTATTTTATACT CAGAGTTCAC CAGGAACAGG AGAGAGGGTC  6801 TCAAATTCTG AATCTAGTTGGCAAGATTCT GAAATCAAGG CATATAATCA AGTTTAAGAC TTAGATCAAC CGTTCTAAGACTTTAGTTCC GTATATTAGT  6851 GTAATAAGTG ATGATAGAAG GGTATATAGA AGAATTTTATTATATGAGAG CATTATTCAC TACTATCTTC CCATATATCT TCTTAAAATA ATATACTCTC  6901GGTGAAATCC CAGCAATTTG GGAGGCTGAG GCAGGAGAAT CGCTTGATCC CCACTTTAGGGTCGTTAAAC CCTCCGACTC CGTCCTCTTA GCGAACTAGG  6951 TGGGAGGCAG AGGTTGCAGTGAGCCAAGAT TGTGCCACTG CATTCCAGCC ACCCTCCGTC TCCAACGTCA CTCGGTTCTAACACGGTGAC GTAAGGTCGG  7001 CAGGTGACAG CATGAGACTC CGTCACAAAA AAAAAAGAAAAAAAAGGGGG GTCCACTGTC GTACTCTGAG GCAGTGTTTT TTTTTTCTTT TTTTTCCCCC  7051GGGGGGGCGG TGGAGCCAAG ATGACCGAAT AGGAACAGCT CCAGTACTAT CCCCCCCGCCACCTCGGTTC TACTGGCTTA TCCTTGTCGA GGTCATGATA  7101 AGCTCCCATC GTGAGTGACGCAGAAGACGG GTGATTTCTG CATTTCCAAC TCGAGGGTAG CACTCACTGC GTCTTCTGCCCACTAAAGAC GTAAAGGTTG  7151 TGAGGTACCA GGTTCATCTC ACAGGGAAGT GCCAGGCAGTGGGTGCAGGA ACTCCATGGT CCAAGTAGAG TGTCCCTTCA CGGTCCGTCA CCCACGTCCT  7201CAGTAGGTGC AGTGCACTGT GCATGAGCCG AAGCAGGGAC GAGGCATCAC GTCATCCACGTCACGTGACA CGTACTCGGC TTCGTCCCTG CTCCGTAGTG  7251 CTCACCCGGG AAGCACAAGGGGTCAGGGAA TTCCCTTTCC TAGTCAAAGA GAGTGGGCCC TTCGTGTTCC CCAGTCCCTTAAGGGAAAGG ATCAGTTTCT  7301 AAAGGGTGAC AGATGGCACC TGGAAAATCG GGTCACTCCCGCCCTAATAC TTTCCCACTG TCTACCGTGG ACCTTTTAGC CCAGTGAGGG CGGGATTATG               HindIII                ~~~~~~~  7351 TGCGCTCTTCCAACAAGCTT GTCTTTGGAA AATAGATCAA TTTCCCTTGG ACGCGAGAAG GTTGTTCGAACAGAAACCTT TTATCTAGTT AAAGGGAACC  7401 GAAGAAGATT TTTAGCACAG CAAGGGGCAGGATGTTCAAC TGTGAGAAAA CTTCTTCTAA AAATCGTGTC GTTCCCCGTC CTACAAGTTGACACTCTTTT  7451 CGAAGAATTA GCCAAAAAAC TTCCAGTAAG CCTGCAAAAA AAAAAAAAAAGCTTCTTAAT CGGTTTTTTG AAGGTCATTC GGACGTTTTT TTTTTTTTTT  7501 ATAAAAGCTAAGTTTCTATA AATGTTCTGT AAATGTAAAA CAGAAGGTAA TATTTTCGAT TCAAAGATATTTACAAGACA TTTACATTTT GTCTTCCATT  7551 GTCAACTGCA CCTAATAAAA ATCACTTAATAGCAATGTGC TGTGTCAGTT CAGTTGACGT GGATTATTTT TAGTGAATTA TCGTTACACGACACAGTCAA  7601 GTTTATTGGA ACCACACCCG GTACACATCC TGTCCAGCAT TTGCAGTGCGCAAATAACCT TGGTGTGGGC CATGTGTAGG ACAGGTCGTA AACGTCACGC  7651 TGCATTGAATTATTGTGCTG GCTAGACTTC ATGGCGCCTG GCACCGAATC ACGTAACTTA ATAACACGACCGATCTGAAG TACCGCGGAC CGTGGCTTAG  7701 CTGCCTTCTC AGCGAAAATG AATAATTGCTTTGTTGGCAA GAAACTAAGC GACGGAAGAG TCGCTTTTAC TTATTAACGA AACAACCGTTCTTTGATTCG  7751 ATCAATGGGA CGCGTGCAAA GCACCGGCGG CGGTAGATGC GGGGTAAGTATAGTTACCCT GCGCACGTTT CGTGGCCGCC GCCATCTACG CCCCATTCAT  7801 CTGAATTTTAATTCGACCTA TCCCGGTAAA GCGAAAGCGA CACGCTTTTT GACTTAAAAT TAAGCTGGATAGGGCCATTT CGCTTTCGCT GTGCGAAAAA  7851 TTTCACACAT AGCGGGACCG AACACGTTATAAGTATCGAT TAGGTCTATT AAAGTGTGTA TCGCCCTGGC TTGTGCAATA TTCATAGCTAATCCAGATAA  7901 TTTGTCTCTC TGTCGGAACC AGAACTGGTA AAAGTTTCCA TTGCGTCTGGAAACAGAGAG ACAGCCTTGG TCTTGACCAT TTTCAAAGGT AACGCAGACC  7951 GCTTGTCTATCATTGCGTCT CTATGGTTTT TGGAGGATTA GACGGGGCCA CGAACAGATA GTAACGCAGAGATACCAAAA ACCTCCTAAT CTGCCCCGGT  8001 CCAGTAATGG TGCATAGCGG ATGTCTGTACCGCCATCGGT GCACCGATAT GGTCATTACC ACGTATCGCC TACAGACATG GCGGTAGCCACGTGGCTATA  8051 AGGTTTGGGG CTCCCCAAGG GACTGCTGGG ATGACAGCTT CATATTATATTCCAAACCCC GAGGGGTTCC CTGACGACCC TACTGTCGAA GTATAATATA  8101 TGAATGGGCGCATAATCAGC TTAATTGGTG AGGACAAGCT ACAAGTTGTA ACTTACCCGC GTATTAGTCGAATTAACCAC TCCTGTTCGA TGTTCAACAT  8151 ACCTGATCTC CACAAAGTAC GTTGCCGGTCGGGGTCAAAC CGTCTTCGGT TGGACTAGAG GTGTTTCATG CAACGGCCAG CCCCAGTTTGGCAGAAGCCA  8201 GCTCGAAACC GCCTTAAACT ACAGACAGGT CCCAGCCAAG TAGGCGGATCCGAGCTTTGG CGGAATTTGA TGTCTGTCCA GGGTCGGTTC ATCCGCCTAG  8251 AAAACCTCAAAAAGGCGGGA GCCAATCAAA ATGCAGCATT ATATTTTAAG TTTTGGAGTT TTTCCGCCCTCGGTTAGTTT TACGTCGTAA TATAAAATTC  8301 CTCACCGAAA CCGGTAAGTA AAGACTATGTATTTTTTCCC AGTGAATAAT GAGTGGCTTT GGCCATTCAT TTCTGATACA TAAAAAAGGGTCACTTATTA  Start E1 SEQ ID NO: 4   MetAlaAsn AspLysGly SerAsnTrp                         ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8351 TGTTGTTAACTATAAAAAGC GTCATGGCAA ACGATAAAGG TAGCAATTGG ACAACAATTG ATATTTTTCGCAGTACCGTT TGCTATTTCC ATCGTTAACC AspSerGlyLeu GlyCysSer TyrLeuLeuThrGluAlaGlu CysGluSer*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8401 GATTCGGGCTTGGGATGCTC ATATCTGCTG ACTGAGGCAG AATGTGAAAG CTAAGCCCGA ACCCTACGAGTATAGACGAC TGACTCCGTC TTACACTTTC *AspLysGlu AsnGluGluPro GlyAlaGlyValGluLeu SerValGluSer*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8451 TGACAAAGAGAATGAGGAAC CCGGGGCAGG TGTAGAACTG TCTGTGGAAT ACTGTTTCTC TTACTCCTTGGGCCCCGTCC ACATCTTGAC AGACACCTTA *SAspArgTyr AspSerGln AspGluAspPheValAspAsn AlaSerVal~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8501 CTGATCGGTATGATAGCCAG GATGAGGATT TTGTTGACAA TGCATCAGTC GACTAGCCAT ACTATCGGTCCTACTCCTAA AACAACTGTT ACGTAGTCAG PheGlnGlyAsn HisLeuGlu ValPheGlnAlaLeuGluLys LysAlaGly*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8551 TTTCAGGGAAATCACCTGGA GGTCTTCCAG GCATTAGAGA AAAAGGCGGG AAAGTCCCTT TAGTGGACCTCCAGAAGGTC CGTAATCTCT TTTTCCGCCC *GluGluGln IleLeuAsnLeu LysArgLysValLeuGly SerSerGlnAsn*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8601 TGAGGAGCAGATTTTAAATT TGAAAAGAAA AGTATTGGGG AGTTCGCAAA ACTCCTCGTC TAAAATTTAAACTTTTCTTT TCATAACCCC TCAAGCGTTT *ASerSerGly SerGluAla SerGluThrProValLysArg ArgLysSer~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8651 ACAGCAGCGGTTCCGAAGCA TCTGAAACTC CAGTTAAAAG ACGGAAATCA TGTCGTCGCC AAGGCTTCGTAGACTTTGAG GTCAATTTTC TGCCTTTAGT GlyAlaLysArg ArgLeuPhe AlaGluAsnGluAlaAsnArg ValLeuThr*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8701 GGAGCAAAGCGAAGATTATT TGCTGAAAAT GAAGCTAACC GTGTTCTTAC CCTCGTTTCG CTTCTAATAAACGACTTTTA CTTCGATTGG CACAAGAATG *ProLeuGln ValGlnGlyGlu GlyGluGlyArgGlnGlu LeuAsnGluGlu*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8751 GCCCCTCCAGGTACAGGGGG AGGGGGAGGG GAGGCAAGAA CTTAATGAGG CGGGGAGGTC CATGTCCCCCTCCCCCTCCC CTCCGTTCTT GAATTACTCC *GGlnAlaIle SerHisLeu HisLeuGlnLeuValLysSer LysAsnAla~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8801 AGCAGGCAATTAGTCATCTA CATCTGCAGC TTGTTAAATC TAAAAATGCT TCGTCCGTTA ATCAGTAGATGTAGACGTCG AACAATTTAG ATTTTTACGA ThrValPheLys LeuGlyLeu PheLysSerLeuPheLeuCys SerPheHis*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8851 ACAGTTTTTAAGCTGGGGCT CTTTAAATCT TTGTTCCTTT GTAGCTTCCA TGTCAAAAAT TCGACCCCGAGAAATTTAGA AACAAGGAAA CATCGAAGGT *AspIleThr ArgLeuPheLys AsnAspLysThrThrAsn GlnGlnTrpVal*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8901 TGATATTACGAGGTTGTTTA AGAATGATAA GACCACTAAT CAGCAATGGG ACTATAATGC TCCAACAAATTCTTACTATT CTGGTGATTA GTCGTTACCC *VLeuAlaVal PheGlyLeu AlaGluValPhePheGluAla SerPheGlu~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  8951 TGCTGGCTGTGTTTGGCCTT GCAGAGGTGT TTTTTGAGGC GAGTTTCGAA ACGACCGACA CAAACCGGAACGTCTCCACA AAAAACTCCG CTCAAAGCTT LeuLeuLysLys GlnCysSer PheLeuGlnMetGlnLysArg SerHisGlu*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9001 CTCCTAAAGAAGCAGTGTAG TTTTCTGCAG ATGCAAAAAA GATCTCATGA GAGGATTTCT TCGTCACATCAAAAGACGTC TACGTTTTTT CTAGAGTACT *GlyGlyThr CysAlaValTyr LeuIleCysPheAsnThr AlaLysSerArg*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9051 AGGAGGAACTTGTGCAGTTT ACTTAATCTG CTTTAACACA GCTAAAAGCA TCCTCCTTGA ACACGTCAAATGAATTAGAC GAAATTGTGT CGATTTTCGT *AGluThrVal ArgAsnLeu MetAlaAsnMetLeuAsnVal ArgGluGlu~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9101 GAGAAACAGTCCGGAATCTG ATGGCAAACA TGCTAAATGT AAGAGAAGAG CTCTTTGTCA GGCCTTAGACTACCGTTTGT ACGATTTACA TTCTCTTCTC CysLeuMetLeu GlnProPro LysIleArgGlyLeuSerAla AlaLeuPhe*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9151 TGTTTGATGCTGCAGCCACC TAAAATTCGA GGACTCAGCG CAGCTCTATT ACAAACTACG ACGTCGGTGGATTTTAAGCT CCTGAGTCGC GTCGAGATAA *TrpPheLys SerSerLeuSer ProAlaThrLeuLysHis GlyAlaLeuPro*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9201 CTGGTTTAAAAGTAGTTTGT CACCCGCTAC ACTTAAACAT GGTGCTTTAC GACCAAATTT TCATCAAACAGTGGGCGATG TGAATTTGTA CCACGAAATG *PGluTrpIle ArgAlaGln ThrThrLeuAsnGluSerLeu GlnThrGlu~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9251 CTGAGTGGATACGGGCGCAA ACTACTCTGA ACGAGAGCTT GCAGACCGAG GACTCACCTA TGCCCGCGTTTGATGAGACT TGCTCTCGAA CGTCTGGCTC LysPheAspPhe GlyThrMet ValGlnTrpAlaTyrAspHis LysTyrAla*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9301 AAATTCGACTTCGGAACTAT GGTGCAATGG GCCTATGATC ACAAATATGC TTTAAGCTGA AGCCTTGATACCACGTTACC CGGATACTAG TGTTTATACG *GluGluSer LysIleAlaTyr GluTyrAlaLeuAlaAla GlySerAspSer*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9351 TGAGGAGTCTAAAATAGCCT ATGAATATGC TTTGGCTGCA GGATCTGATA ACTCCTCAGA TTTTATCGGATACTTATACG AAACCGACGT CCTAGACTAT *SAsnAlaArg AlaPheLeu AlaThrAsnSerGlnAlaLys HisValLys~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9401 GCAATGCACGGGCTTTTTTA GCAACTAACA GCCAAGCTAA GCATGTGAAG CGTTACGTGC CCGAAAAAATCGTTGATTGT CGGTTCGATT CGTACACTTC AspCysAlaThr MetValArg HisTyrLeuArgAlaGluThr GlnAlaLeu*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9451 GACTGTGCAACTATGGTAAG ACACTATCTA AGAGCTGAAA CACAAGCATT CTGACACGTT GATACCATTCTGTGATAGAT TCTCGACTTT GTGTTCGTAA *SerMetPro AlaTyrIleLys AlaArgCysLysLeuAla ThrGlyGluGly*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9501 AAGCATGCCTGCATATATTA AAGCTAGGTG CAAGCTGGCA ACTGGGGAAG TTCGTACGGA CGTATATAATTTCGATCCAC GTTCGACCGT TGACCCCTTC *GSerTrpLys SerIleLeu ThrPhePheAsnTyrGlnAsn IleGluLeu~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9551 GAAGCTGGAAGTCTATCCTA ACTTTTTTTA ACTATCAGAA TATTGAATTA CTTCGACCTT CAGATAGGATTGAAAAAAAT TGATAGTCTT ATAACTTAAT IleThrPheIle AsnAlaLeu LysLeuTrpLeuLysGlyIle ProLysLys*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9601 ATTACCTTTATTAATGCTTT AAAGCTCTGG CTAAAAGGAA TTCCAAAAAA TAATGGAAAT AATTACGAAATTTCGAGACC GATTTTCCTT AAGGTTTTTT *AsnCysLeu AlaPheIleGly ProProAsnThrGlyLys SerMetLeuCys*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9651 AAACTGTTTAGCATTTATTG GCCCTCCAAA CACAGGCAAG TCTATGCTCT TTTGACAAAT CGTAAATAACCGGGAGGTTT GTGTCCGTTC AGATACGAGA *CAsnSerLeu IleHisPhe LeuGlyGlySerValLeuSer PheAlaAsn~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9701 GCAACTCATTAATTCATTTT TTGGGTGGTA GTGTTTTATC TTTTGCCAAC CGTTGAGTAA TTAAGTAAAAAACCCACCAT CACAAAATAG AAAACGGTTG HisLysSerHis PheTrpLeu AlaSerLeuAlaAspThrArg AlaAlaLeu*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9751 CATAAAAGTCACTTTTGGCT TGCTTCCCTA GCAGATACTA GAGCTGCTTT GTATTTTCAG TGAAAACCGAACGAAGGGAT CGTCTATGAT CTCGACGAAA *ValAspAsp AlaThrHisAla CysTrpArgTyrPheAsp ThrTyrLeuArg*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9801 AGTAGATGATGCTACTCATG CTTGCTGGAG GTACTTTGAC ACATACCTCA TCATCTACTA CGATGAGTACGAACGACCTC CATGAAACTG TGTATGGAGT *AAsnAlaLeu AspGlyTyr ProValSerIleAspArgLys HisLysAla~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9851 GAAATGCATTGGATGGCTAC CCTGTCAGTA TTGATAGAAA ACACAAAGCA CTTTACGTAA CCTACCGATGGGACAGTCAT AACTATCTTT TGTGTTTCGT AlaValGlnIle LysAlaPro ProLeuLeuValThrSerAsn IleAspVal*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9901 GCGGTTCAAATTAAAGCTCC ACCCCTCCTG GTAACCAGTA ATATTGATGT CGCCAAGTTT AATTTCGAGGTGGGGAGGAC CATTGGTCAT TATAACTACA *GlnAlaGlu AspArgTyrLeu TyrLeuHisSerArgVal GlnThrPheArg*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~  9951 GCAGGCAGAGGACAGATATT TGTACTTGCA TAGTCGGGTG CAAACCTTTC CGTCCGTCTC CTGTCTATAAACATGAACGT ATCAGCCCAC GTTTGGAAAG *APheGluGln ProCysThr AspGluSerGlyGluGlnPro PheAsnIle~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10001 GCTTTGAGCAGCCATGCACA GATGAATCGG GTGAGCAACC TTTTAATATT CGAAACTCGT CGGTACGTGTCTACTTAGCC CACTCGTTGG AAAATTATAA ThrAspAlaAsp TrpLysSer PhePheValArgLeuTrpGly ArgLeuAsp*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10051 ACTGATGCAGATTGGAAATC TTTTTTTGTA AGGTTATGGG GGCGTTTAGA TGACTACGTC TAACCTTTAGAAAAAAACAT TCCAATACCC CCGCAAATCT              SEQ ID NO: 5 Start E2MetGluThr AlaCysGlu                                   ~~~~~~~~~~~~~~~~~~~ *LeuIleAspGluGluGluAsp SerGluGlu AspGlyAsp SerMetArgThr*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10101 CCTGATTGACGAGGAGGAGG ATAGTGAAGA GGATGGAGAC AGCATGCGAA GGACTAACTG CTCCTCCTCCTATCACTTCT CCTACCTCTG TCGTACGCTT ArgLeuHisAla AlaGlnGlu ThrGlnMetGlnLeuIleGlu LysSerSer*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *TPheThrCysSerAlaArg AsnThrAsnAla ValAsp Stop E1~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10151 CGTTTACATG CAGCGCAAGAAACACAAATG CAGTTGATTG AGAAAAGTAG GCAAATGTAC GTCGCGTTCT TTGTGTTTACGTCAACTAAC TCTTTTCATC *AspLysLeu GlnAspHisIle LeuTyrTrp ThrAlaValArgThrGluAsn* ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~10201 TGATAAGTTG CAAGATCATA TACTGTACTG GACTGCTGTT AGAACTGAGA ACTATTCAACGTTCTAGTAT ATGACATGAC CTGACGACAA TCTTGACTCT *AThrLeuLeu TyrAlaAlaArgLysLysGly ValThrVal LeuGlyHis~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10251 ACACACTGCTTTATGCTGCA AGGAAAAAAG GGGTGACTGT CCTAGGACAC TGTGTGACGA AATACGACGTTCCTTTTTTC CCCACTGACA GGATCCTGTG CysArgValPro HisSerVal ValCysGlnGluArgAlaLys GlnAlaIle*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10301 TGCAGAGTACCACACTCTGT AGTTTGTCAA GAGAGAGCCA AGCAGGCCAT ACGTCTCATG GTGTGAGACATCAAACAGTT CTCTCTCGGT TCGTCCGGTA *GluMetGln LeuSerLeuGln GluLeuSerLysThrGlu PheGlyAspGlu*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10351 TGAAATGCAGTTGTCTTTGC AGGAGTTAAG CAAAACTGAG TTTGGGGATG ACTTTACGTC AACAGAAACGTCCTCAATTC GTTTTGACTC AAACCCCTAC *GProTrpSer LeuLeuAsp ThrSerTrpAspArgTyrMet SerGluPro~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10401 AACCATGGTCTTTGCTTGAC ACAAGCTGGG ACCGATATAT GTCAGAACCT TTGGTACCAG AAACGAACTGTGTTCGACCC TGGCTATATA CAGTCTTGGA LysArgCysPhe LysLysGly AlaArgValValGluValGlu PheAspGly*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10451 AAACGGTGCTTTAAGAAAGG CGCCAGGGTG GTAGAGGTGG AGTTTGATGG TTTGCCACGA AATTCTTTCCGCGGTCCCAC CATCTCCACC TCAAACTACC *AsnAlaSer AsnThrAsnTrp TyrThrValTyrSerAsn LeuTyrMetArg*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10501 AAATGCAAGCAATACAAACT GGTACACTGT CTACAGCAAT TTGTACATGC TTTACGTTCG TTATGTTTGACCATGTGACA GATGTCGTTA AACATGTACG *AThrGluAsp GlyTrpGln LeuAlaLysAlaGlyLeuThr GluLeuGly~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10551 GCACAGAGGACGGCTGGCAG CTTGCGAAGG CTGGGCTGAC GGAACTGGGC CGTGTCTCCT GCCGACCGTCGAACGCTTCC GACCCGACTG CCTTGACCCG SerThrThrAla ProTrpPro ValLeuAspAlaPheThrIle LeuAlaLeu*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 10601 TCTACTACTGCACCATGGCC GGTGCTGGAC GCATTTACTA TTCTCGCTTT AGATGATGAC GTGGTACCGGCCACGACCTG CGTAAATGAT AAGAGCGAAA *ValThrArg GlnProAspLeu ValGlnGlnGlyIleThr Leu Stop E2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~10651 GGTGACGAGG CAGCCAGATT TAGTACAACA GGGCATTACT CTGTAAGAGA CCACTGCTCCGTCGGTCTAA ATCATGTTGT CCCGTAATGA GACATTCTCT 10701 TCAGGACAGA GTGTATGCTGGTGTCTCATC CACCTCTTCT GATTTTAGAG AGTCCTGTCT CACATACGAC CACAGAGTAGGTGGAGAAGA CTAAAATCTC 10751 ATCGCCCAGA CGGAGTCTGG GTCGCATCCG AAGGACCTGAAGGAGACCCT TAGCGGGTCT GCCTCAGACC CAGCGTAGGC TTCCTGGACT TCCTCTGGGA 10801GCAGGAAAAG AAGCCGAGCC AGCCCAGCCT GTCTCTTCTT TGCTCGGCTC CGTCCTTTTCTTCGGCTCGG TCGGGTCGGA CAGAGAAGAA ACGAGCCGAG 10851 CCCCGCCTGC GGTCCCATCAGAGCAGGCCT CGGTTGGGTA CGGGACGGTC GGGGCGGACG CCAGGGTAGT CTCGTCCGGAGCCAACCCAT GCCCTGCCAG 10901 CTCGCTCGCA CCCCTACAAT TTTCCTGCAG GCTCGGGGGGCTCTATTCTC GAGCGAGCGT GGGGATGTTA AAAGGACGTC CGAGCCCCCC GAGATAAGAG 10951CGCTCTTCCT CCACCCCGTG CAGGGCACGG TACCGGTGGA CTTGGCATCA GCGAGAAGGAGGTGGGGCAC GTCCCGTGCC ATGGCCACCT GAACCGTAGT 11001 AGGCAGGAAG AAGAGGAGCAGTCGCCCGAC TCCACAGAGG AAGAACCAGT TCCGTCCTTC TTCTCCTCGT CAGCGGGCTGAGGTGTCTCC TTCTTGGTCA 11051 GACTCTCCCA AGGCGCACCA CCAATGATGG ATTCCACCTGTTAAAGGCAG CTGAGAGGGT TCCGCGTGGT GGTTACTACC TAAGGTGGAC AATTTCCGTC 11101GAGGGTCATG CTTTGCTCTA ATTTCAGGAA CTGCTAACCA GGTAAAGTGC CTCCCAGTACGAAACGAGAT TAAAGTCCTT GACGATTGGT CCATTTCACG 11151 TATCGCTTTC GGGTGAAAAAGAACCATAGA CATCGCTACG AGAACTGCAC ATAGCGAAAG CCCACTTTTT CTTGGTATCTGTAGCGATGC TCTTGACGTG 11201 CACCACCTGG TTCACAGTTG CTGACAACGG TGCTGAAAGACAAGGACAAG GTGGTGGACC AAGTGTCAAC GACTGTTGCC ACGACTTTCT GTTCCTGTTC 11251CACAAATACT GATCACCTTT GGATCGCCAA GTCAAAGGCA AGACTTTCTG GTGTTTATGACTAGTGGAAA CCTAGCGGTT CAGTTTCCGT TCTGAAAGAC 11301 AAACATGTAC CACTACCTCCTGGAATGAAC ATTTCCGGCT TTACAGCCAG TTTGTACATG GTGATGGAGG ACCTTACTTGTAAAGGCCGA AATGTCGGTC 11351 CTTGGACTTC TGATCACTGC CATTGCCTTT TCTTCATCTGACTGGTGTAC GAACCTGAAG ACTAGTGACG GTAACGGAAA AGAAGTAGAC TGACCACATG 11401TATGCCAAAT CTATGCGACC GCATTATAAA GCCGAATTCT GCAGATATCC ATACGGTTTAGATACGCTGG CGTAATATTT CGGCTTAAGA CGTCTATAGG 11451 ATCACACTGG CGGCCATATGGCCGCTATGC GGTGTGAAAT ACCGCACAGA TAGTGTGACC GCCGGTATAC CGGCGATACGCCACACTTTA TGGCGTGTCT 11501 TGCGTAAGGA GAAAATACCG CATCAGGCGC TCTTCCGCTTCCTCGCTCAC ACGCATTCCT CTTTTATGGC GTAGTCCGCG AGAAGGCGAA GGAGCGAGTG 11551TGACTCGCTG CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT ACTGAGCGACGCGAGCCAGC AAGCCGACGC CGCTCGCCAT AGTCGAGTGA 11601 CAAAGGCGGT AATACGGTTATCCACAGAAT CAGGGGATAA CGCAGGAAAG GTTTCCGCCA TTATGCCAAT AGGTGTCTTAGTCCCCTATT GCGTCCTTTC 11651 AACATGTGAG CAAAAGGCCA GCAAAAGGCC AGGAACCGTAAAAAGGCCGC TTGTACACTC GTTTTCCGGT CGTTTTCCGG TCCTTGGCAT TTTTCCGGCG 11701GTTGCTGGCG TTTTTCCATA GGCTCCGCCC CCCTGACGAG CATCACAAAA CAACGACCGCAAAAAGGTAT CCGAGGCGGG GGGACTGCTC GTAGTGTTTT 11751 ATCGACGCTC AAGTCAGAGGTGGCGAAACC CGACAGGACT ATAAAGATAC TAGCTGCGAG TTCAGTCTCC ACCGCTTTGGGCTGTCCTGA TATTTCTATG 11801 CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTGTTCCGACCCT GTCCGCAAAG GGGGACCTTC GAGGGAGCAC GCGAGAGGAC AAGGCTGGGA 11851GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC CGGCGAATGGCCTATGGACA GGCGGAAAGA GGGAAGCCCT TCGCACCGCG 11901 TTTCTCATAG CTCACGCTGTAGGTATCTCA GTTCGGTGTA GGTCGTTCGC AAAGAGTATC GAGTGCGACA TCCATAGAGTCAAGCCACAT CCAGCAAGCG 11951 TCCAAGCTGG GCTGTGTGCA CGAACCCCCC GTTCAGCCCGACCGCTGCGC AGGTTCGACC CGACACACGT GCTTGGGGGG CAAGTCGGGC TGGCGACGCG 12001CTTATCCGGT AACTATCGTC TTGAGTCCAA CCCGGTAAGA CACGACTTAT GAATAGGCCATTGATAGCAG AACTCAGGTT GGGCCATTCT GTGCTGAATA 12051 CGCCACTGGC AGCAGCCACTGGTAACAGGA TTAGCAGAGC GAGGTATGTA GCGGTGACCG TCGTCGGTGA CCATTGTCCTAATCGTCTCG CTCCATACAT 12101 GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACGGCTACACTAG CCGCCACGAT GTCTCAAGAA CTTCACCACC GGATTGATGC CGATGTGATC 12151AAGGACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA TTCCTGTCATAAACCATAGA CGCGAGACGA CTTCGGTCAA TGGAAGCCTT 12201 AAAGAGTTGG TAGCTCTTGATCCGGCAAAC AAACCACCGC TGGTAGCGGT TTTCTCAACC ATCGAGAACT AGGCCGTTTGTTTGGTGGCG ACCATCGCCA 12251 GGTTTTTTTG TTTGCAAGCA GCAGATTACG CGCAGAAAAAAAGGATCTCA CCAAAAAAAC AAACGTTCGT CGTCTAATGC GCGTCTTTTT TTCCTAGAGT 12301AGAAGATCCT TTGATCTTTT CTACGGGGTC TGACGCTCAG TGGAACGAAA TCTTCTAGGAAACTAGAAAA GATGCCCCAG ACTGCGAGTC ACCTTGCTTT 12351 ACTCACGTTA AGGGATTTTGGTCATGAGAT TATCAAAAAG GATCTTCACC TGAGTGCAAT TCCCTAAAAC CAGTACTCTAATAGTTTTTC CTAGAAGTGG 12401 TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCTAAAGTATATA ATCTAGGAAA ATTTAATTTT TACTTCAAAA TTTAGTTAGA TTTCATATAT 12451TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA ACTCATTTGAACCAGACTGT CAATGGTTAC GAATTAGTCA CTCCGTGGAT       Stop SEQ ID NO:6  TrpHis LysIleLeuSer AlaGlyIle*                         ***~~~~~~~~~~~~~~~~~~~~~~~~~~ 12501 TCTCAGCGATCTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC AGAGTCGCTA GACAGATAAAGCAAGTAGGT ATCAACGGAC TGAGGGGCAG _(*)GluAlaIle GlnArgAsnArg GluAspMetThrAlaGln SerGlyThrThr*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12551 GTGTAGATAACTACGATACG GGAGGGCTTA CCATCTGGCC CCAGTGCTGC CACATCTATT GATGCTATGCCCTCCCGAAT GGTAGACCGG GGTCACGACG _(*) _(*)TyrIleVal ValIleArgSerProLysGly AspProGly LeuAlaAla~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12601 AATGATACCGCGAGACCCAC GCTCACCGGC TCCAGATTTA TCAGCAATAA TTACTATGGC GCTCTGGGTGCGAGTGGCCG AGGTCTAAAT AGTCGTTATT IleIleGlyArg SerGlyArg GluGlyAlaGlySerLysAsp AlaIlePhe*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12651 ACCAGCCAGCCGGAAGGGCC GAGCGCAGAA GTGGTCCTGC AACTTTATCC TGGTCGGTCG GCCTTCCCGGCTCGCGTCTT CACCAGGACG TTGAAATAGG _(*)TrpGlyAla ProLeuAlaSer ArgLeuLeuProGlyAla ValLysAspAla*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12701 GCCTCCATCCAGTCTATTAA TTGTTGCCGG GAAGCTAGAG TAAGTAGTTC CGGAGGTAGG TCAGATAATTAACAACGGCC CTTCGATCTC ATTCATCAAG _(*) _(*)GluMetTrp AspIleLeuGlnGlnArgSer AlaLeuThr LeuLeuGlu~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12751 GCCAGTTAATAGTTTGCGCA ACGTTGTTGC CATTGCTGCA GGCATCGTGG CGGTCAATTA TCAAACGCGTTGCAACAACG GTAACGACGT CCGTAGCACC GlyThrLeuLeu LysArgLeu ThrThrAlaMetAlaAlaPro MetThrThr*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12801 TGTCACGCTCGTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGA ACAGTGCGAG CAGCAAACCATACCGAAGTA AGTCGAGGCC AAGGGTTGCT _(*)AspArgGlu AspAsnProIle AlaGluAsnLeuGluPro GluTrpArgAsp*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12851 TCAAGGCGAGTTACATGATC CCCCATGTTG TGCAAAAAAG CGGTTAGCTC AGTTCCGCTC AATGTACTAGGGGGTACAAC ACGTTTTTTC GCCAATCGAG _(*) _(*)LeuArgThr ValHisAspGlyMetAsnHis LeuPheAla ThrLeuGlu~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12901 CTTCGGTCCTCCGATCGTTG TCAGAAGTAA GTTGGCCGCA GTGTTATCAC GAAGCCAGGA GGCTAGCAACAGTCTTCATT CAACCGGCGT CACAATAGTG LysProGlyGly IleThrThr LeuLeuLeuAsnAlaAlaThr AsnAspSer*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12951 TCATGGTTATGGCAGCACTG CATAATTCTC TTACTGTCAT GCCATCCGTA AGTACCAATA CCGTCGTGACGTATTAAGAG AATGACAGTA CGGTAGGCAT _(*)MetThrIle AlaAlaSerCys LeuGluArgValThrMet GlyAspThrLeu*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13001 AGATGCTTTTCTGTGACTGG TGAGTACTCA ACCAAGTCAT TCTGAGAATA TCTACGAAAA GACACTGACCACTCATGAGT TGGTTCAGTA AGACTCTTAT _(*) _(*)HisLysGlu ThrValProSerTyrGluVal LeuAspAsn GlnSerTyr~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13051 GTGTATGCGGCGACCGAGTT GCTCTTGCCC GGCGTCAACA CGGGATAATA CACATACGCC GCTGGCTCAACGAGAACGGG CCGCAGTTGT GCCCTATTAT HisIleArgArg GlyLeuGln GluGlnGlyAlaAspValArg SerLeuVal*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13101 CCGCGCCACATAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCT GGCGCGGTGT ATCGTCTTGAAATTTTCACG AGTAGTAACC TTTTGCAAGA _(*)AlaGlyCys LeuLeuValLys PheThrSerMetMetPro PheArgGluGlu*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13151 TCGGGGCGAAAACTCTCAAG GATCTTACCG CTGTTGAGAT CCAGTTCGAT AGCCCCGCTT TTGAGAGTTCCTAGAATGGC GACAACTCTA GGTCAAGCTA _(*) _(*)ProArgPhe SerGluLeuIleLysGlySer AsnLeuAsp LeuGluIle~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13201 GTAACCCACTCGTGCACCCA ACTGATCTTC AGCATCTTTT ACTTTCACCA CATTGGGTGA GCACGTGGGTTGACTAGAAG TCGTAGAAAA TGAAAGTGGT TyrGlyValArg AlaGlyLeu GlnAspGluAlaAspLysVal LysValLeu*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13251 GCGTTTCTGGGTGAGCAAAA ACAGGAAGGC AAAATGCCGC AAAAAAGGGA CGCAAAGACC CACTCGTTTTTGTCCTTCCG TTTTACGGCG TTTTTTCCCT _(*)ThrGluPro HisAlaPheVal ProLeuCysPheAlaAla PhePheProIle*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13301 ATAAGGGCGACACGGAAATG TTGAATACTC ATACTCTTCC TTTTTCAATA TATTCCCGCT GTGCCTTTACAACTTATGAG TATGAGAAGG AAAAAGTTAT _(*) _(*) LeuAlaVal ArgPheHisGlnIleSerMet Start Beta-Lactamase ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~13351 TTATTGAAGC ATTTATCAGG GTTATTGTCT CATGAGCGGA TACATATTTG AATAACTTCGTAAATAGTCC CAATAACAGA GTACTCGCCT ATGTATAAAC 13401 AATGTATTTA GAAAAATAAACAAATAGGGG TTCCGCGCAC ATTTCCCCGA TTACATAAAT CTTTTTATTT GTTTATCCCCAAGGCGCGTG TAAAGGGGCT 13451 AAAGTGCCAC CTGACGTCTA AGAAACCATT ATTATCATGACATTAACCTA TTTCACGGTG GACTGCAGAT TCTTTGGTAA TAATAGTACT GTAATTGGAT 13501TAAAAATAGG CGTATCACGA GGCCCTTTCG TCTTCAAGAA TTCTCATGTT ATTTTTATCCGCATAGTGCT CCGGGAAAGC AGAAGTTCTT AAGAGTACAA                    HindIII                   ~~~~~~~ 13551 TGACAGCTTA TCATCGATAA GCTTCACGCTGCCGCAAGCA CTCAGGGCGC ACTGTCGAAT AGTAGCTATT CGAAGTGCGA CGGCGTTCGTGAGTCCCGCG 13601 AAGGGCTGCT AAAGGAAGCG GAACACGTAG AAAGCCAGTC CGCAGAAACGTTCCCGACGA TTTCCTTCGC CTTGTGCATC TTTCGGTCAG GCGTCTTTGC 13651 GTGCTGACCCCGGATGAATG TCAGCTACTG GGCTATCTGG ACAAGGGAAA CACGACTGGG GCCTACTTACAGTCGATGAC CCGATAGACC TGTTCCCTTT 13701 ACGCAAGCGC AAAGAGAAAG CAGGTAGCTTGCAGTGGGCT TACATGGCGA TGCGTTCGCG TTTCTCTTTC GTCCATCGAA CGTCACCCGAATGTACCGCT 13751 TAGCTAGACT GGGCGGTTTT ATGGACAGCA AGCGAACCGG AATTGCCAGCATCGATCTGA CCCGCCAAAA TACCTGTCGT TCGCTTGGCC TTAACGGTCG 13801 TGGGGCGCCCTCTGGTAAGG TTGGGAAGCC CTGCAAAGTA AACTGGATGG ACCCCGCGGG AGACCATTCCAACCCTTCGG GACGTTTCAT TTGACCTACC 13851 CTTTCTTGCC GCCAAGGATC TGATGGCGCAGGGGATCAAG ATCCTGCTTC GAAAGAACGG CGGTTCCTAG ACTACCGCGT CCCCTAGTTCTAGGACGAAG 13901 ATCCCCGTGG CCCGTTGCTC GCGTTTGCTG GCGGTGTCCC CGGAAGAAATTAGGGGCACC GGGCAACGAG CGCAAACGAC CGCCACAGGG GCCTTCTTTA 13951 ATATTTGCATGTCTTTAGTT CTATGATGAC ACAAACCCCG CCCAGCGTCT TATAAACGTA CAGAAATCAAGATACTACTG TGTTTGGGGC GGGTCGCAGA 14001 TGTCATTGGC GAATTCGAAC ACGCAGATGCAGTCGGGGCG GCGCGGTCCC ACAGTAACCG CTTAAGCTTG TGCGTCTACG TCAGCCCCGCCGCGCCAGGG 14051 AGGTCCACTT CGCATATTAA GGTGACGCGT GTGGCCTCGA ACACCGAGCGTCCAGGTGAA GCGTATAATT CCACTGCGCA CACCGGAGCT TGTGGCTCGC 14101 ACCCTGCAGCGACCCGCTTA ACAGCGTCAA CAGCGTGCCG CAGATCTGAT TGGGACGTCG CTGGGCGAATTGTCGCAGTT GTCGCACGGC GTCTAGACTA Start  G418 resist SEQ ID NO:7      Met IleGluGlnAsp GlyLeuHis*                                     ~~~~~~~~~~~~~~~~~~~~~~~~~ 14151CAAGAGACAG GATGAGGATC GTTTCGCATG ATTGAACAAG ATGGATTGCA GTTCTCTGTCCTACTCCTAG CAAAGCGTAC TAACTTGTTC TACCTAACGT *AlaGlySer ProAlaAlaTrpValGluArg LeuPheGly TyrAspTrpAla*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14201 CGCAGGTTCTCCGGCCGCTT GGGTGGAGAG GCTATTCGGC TATGACTGGG GCGTCCAAGA GGCCGGCGAACCCACCTCTC CGATAAGCCG ATACTGACCC *AGlnGlnThr IleGlyCys SerAspAlaAlaValPheArg LeuSerAla~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14251 CACAACAGACAATCGGCTGC TCTGATGCCG CCGTGTTCCG GCTGTCAGCG GTGTTGTCTG TTAGCCGACGAGACTACGGC GGCACAAGGC CGACAGTCGC GlnGlyArgPro ValLeuPhe ValLysThrAspLeuSerGly AlaLeuAsn*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14301 CAGGGGCGCCCGGTTCTTTT TGTCAAGACC GACCTGTCCG GTGCCCTGAA GTCCCCGCGG GCCAAGAAAAACAGTTCTGG CTGGACAGGC CACGGGACTT *GluLeuGln AspGluAlaAla ArgLeuSerTrpLeuAla ThrThrGlyVal*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14351 TGAACTGCAGGACGAGGCAG CGCGGCTATC GTGGCTGGCC ACGACGGGCG ACTTGACGTC CTGCTCCGTCGCGCCGATAG CACCGACCGG TGCTGCCCGC *VProCysAla AlaValLeu AspValValThrGluAlaGly ArgAspTrp~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14401 TTCCTTGCGCAGCTGTGCTC GACGTTGTCA CTGAAGCGGG AAGGGACTGG AAGGAACGCG TCGACACGAGCTGCAACAGT GACTTCGCCC TTCCCTGACC LeuLeuLeuGly GluValPro GlyGlnAspLeuLeuSerSer HisLeuAla*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14451 CTGCTATTGGGCGAAGTGCC GGGGCAGGAT CTCCTGTCAT CTCACCTTGC GACGATAACC CGCTTCACGGCCCCGTCCTA GAGGACACTA GAGTGGAACG                                          ~~~~~~~~ *ProAlaGluLysValSerIle MetAlaAsp AlaMetArg ArgLeuHisThr*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14501 TCCTGCCGAGAAAGTATCCA TCATGGCTGA TGCAATGCGG CGGCTGCATA AGGACGGCTC TTTCATAGGTACTACCGACT ACGTTACGCC GCCGACGTAT~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *TLeuAspProAlaThrCys ProPheAspHis GlnAlaLys HisArgIle~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14551 CGCTTGATCCGGCTACCTGC CCATTCGACC ACCAAGCGAA ACATCGCATC GCGAACTAGG CCGATGGACGGGTAAGCTGG TGGTTCGCTT TGTAGCGTAG~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ GluArgAlaArgThrArgMet GluAlaGly LeuValAspGln AspAspLeu*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14601 GAGCGAGCACGTACTCGGAT GGAAGCCGGT CTTGTCGATC AGGATGATCT CTCGCTCGTG CATGAGCCTACCTTCGGCCA GAACAGCTAG TCCTACTAGA~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *AspGluGluHisGlnGlyLeu AlaProAla GluLeuPhe AlaArgLeuLys*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14651 GGACGAAGAGCATCAGGGGC TCGCGCCAGC CGAACTGTTC GCCAGGCTCA CCTGCTTCTC GTAGTCCCCGAGCGCGGTCG GCTTGACAAG CGGTCCGAGT~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *LAlaArgMetProAspGly GluAspLeuVal ValThrHis GlyAspAla~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14701 AGGCGCGCATGCCCGACGGC GAGGATCTCG TCGTGACCCA TGGCGATGCC TCCGCGCGTA CGGGCTGCCGCTCCTAGAGC AGCACTGGGT ACCGCTACGG~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ CysLeuProAsnIleMetVal GluAsnGly ArgPheSerGly PheIleAsp*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14751 TGCTTGCCGAATATCATGGT GGAAAATGGC CGCTTTTCTG GATTCATCGA ACGAACGGCT TATAGTACCACCTTTTACCG GCGAAAAGAC CTAAGTAGCT~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *CysGlyArgLeuGlyValAla AspArgTyr GlnAspIle AlaLeuAlaThr*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14801 CTGTGGCCGGCTGGGTGTGG CGGACCGCTA TCAGGACATA GCGTTGGCTA GACACCGGCC GACCCACACCGCCTGGCGAT AGTCCTGTAT CGCAACCGAT~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *TArgAspIleAlaGluGlu LeuGlyGlyGlu TrpAlaAsp ArgPheLeu~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14851 CCCGTGATATTGCTGAAGAG CTTGGCGGCG AATGGGCTGA CCGCTTCCTC GGGCACTATA ACGACTTCTCGAACCGCCGC TTACCCGACT GGCGAAGGAG~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ValLeuTyrGlyIleAlaAla ProAspSer GlnArgIleAla PheTyrArg*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14901 GTGCTTTACGGTATCGCCGC TCCCGATTCG CAGCGCATCG CCTTCTATCG CACGAAATGC CATAGCGGCGAGGGCTAAGC GTCGCGTAGC GGAAGATAGC~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ *LeuLeuAspGluPhePhe*** Stop ~~~~~~~~~~~~~~~~~~~~ 14951 CCTTCTTGAC GAGTTCTTCTGAGCGGGACT CTGGGGTTCG AAATGACCGA GGAAGAACTG CTCAAGAAGA CTCGCCCTGAGACCCCAAGC TTTACTGGCT 15001 CCAAGCGACG CCCAACCTGC CATCACGAGA TTTCGATTCCACCGCCGCCT GGTTCGCTGC GGGTTGGACG GTAGTGCTCT AAAGCTAAGG TGGCGGCGGA 15051TCTATGAAAG GTTGGGCTTC GGAATCGTTT TCCGGGACGC CGGCTGGATG AGATACTTTCCAACCCGAAG CCTTAGCAAA AGGCCCTGCG GCCGACCTAC 15101 ATCCTCCAGC GCGGGGATCTCATGCTGGAG TTCTTCGCCC ACCCCGGGAG TAGGAGGTCG CGCCCCTAGA GTACGACCTCAAGAAGCGGG TGGGGCCCTC 15151 ATGGGGGAGG CTAACTGAAA CACGGAAGGA GACAATACCGGAAGGAACCC TACCCCCTCC GATTGACTTT GTGCCTTCCT CTGTTATGGC CTTCCTTGGG 15201GCGCTATGAA CGGCAATAAA AAGACAGAAT AAAACGCACG GTGTTGGGTC CGCGATACTTGCCGTTATTT TTCTGTCTTA TTTTGCGTGC CACAACCCAG~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 15251 GTTTGTTCATAAACGCGGGG TTCGGTCCCA GGGCTGGCAC TCTGTCGATA CAAACAAGTA TTTGCGCCCCAAGCCAGGGT CCCGACCGTG AGACAGCTAT~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 15301 CCCCACCGAGACCCCATTGG GGCCAATACG CCCGCGTTTC TTCCTTTTCC GGGGTGGCTC TGGGGTAACCCCGGTTATGC GGGCGCAAAG AAGGAAAAGG~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 15351 CCACCCCACCCCCCAAGTTC GGGTGAAGGC CCAGGGCTCG CAGCCAACGT GGTGGGGTGG GGGGTTCAAGCCCACTTCCG GGTCCCGAGC GTCGGTTGCA~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 15401 CGGGGCGGCAAGCCCTGCCA TAGCCACGGG CCCCGTGGGT TAGGGACGGC GCCCCGCCGT TCGGGACGGTATCGGTGCCC GGGGCACCCA ATCCCTGCCG~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 15451  GGATCGCGGCCC        CCTAGCGCCG GG       ~~~~~~~~~~~~~

1. A method of producing a protective immune response in a human subjectcomprising administering to the subject an effective amount of lungcancer cells transfected with a eukaryotic expression vector derivedfrom the bovine papilloma virus comprising a nucleic acid encoding CD80(B7.1) and with a eukaryotic expression vector derived from the bovinepapilloma virus comprising a nucleic acid encoding an HLA antigen. 2.The method of claim 1, wherein the eukaryotic expression vectorcomprises a nucleic acid encoding a mouse metallothionein-I promoter(MTI).
 3. The method of claim 1, wherein the eukaryotic expressionvector comprises a nucleic acid encoding a G418 resistance gene orhistidinol resistance gene.
 4. The method of claim 1, wherein the HLAantigen is selected from HLA A1, HLA A2, HLA A3 and HLA A27.
 5. Themethod of claim 1, wherein the lung cancer cell is an adenocarcinoma. 6.The method of claim 2, wherein the HLA antigen is selected from HLA A1,HLA A2, HLA A3 and HLA A27.
 7. The method of claim 2, wherein the lungcancer cell is an adenocarcinoma.
 8. The method of claim 1, wherein thelung cancer cells are administered more than once.
 9. A method ofproducing a protective immune response in a human subject comprisingadministering to the subject an effective amount of lung cancer cellstransfected with a eukaryotic expression vector derived from the bovinepapilloma virus comprising a nucleic acid encoding CD80 (B7.1) and anucleic acid encoding an HLA antigen.
 10. The method of claim 9, whereinthe eukaryotic expression vector comprises a nucleic acid encoding amouse metallothionein-I promoter (MTI) and a nucleic acid encoding acytomegalovirus (CMV) promoter.
 11. The method of claim 9, wherein theeukaryotic expression vector comprises a nucleic acid encoding a G418resistance gene or histidinol resistance gene.
 12. The method of claim9, wherein the HLA antigen is selected from HLA A1, HLA A2, HLA A3 andHLA A27.
 13. The method of claim 9, wherein the lung cancer cell is anadenocarcinoma.
 14. The method of claim 10, wherein the HLA antigen isselected from HLA A1, HLA A2, HLA A3 and HLA A27.
 15. The method ofclaim 10, wherein the lung cancer cell is an adenocarcinoma.
 16. Themethod of claim 9, wherein the lung cancer cells are administered morethan once.
 17. A population of lung cancer cells genetically modified toexpress a nucleic acid encoding CD80 (B7.1) and a nucleic acid encodingan HLA antigen, wherein at least at least 70% of the cells co-expressCD80 (B7.1) and an HLA antigen.
 18. The population of lung cancer cellsof claim 17, wherein the HLA antigen is selected from HLA A1, HLA A2,HLA A3 and HLA A27.
 19. The population of lung cancer cells of claim 17,wherein the lung cancer cell is an adenocarcinoma.
 20. A method ofmanufacturing a vaccine against lung cancer comprising geneticallymodifying a population of lung cancer cells to express a nucleic acidencoding CD80 (B7.1) and a nucleic acid encoding an HLA antigen, whereinat least at least 70% of the cells co-express CD80 (B7.1) and an HLAantigen.
 21. The method of claim 20, wherein the HLA antigen is selectedfrom HLA A1, HLA A2, HLA A3 and HLA A27.
 22. The method of claim 20,wherein the lung cancer cell is an adenocarcinoma.